Embryonic or stem-like cell lines produced by cross species nuclear transplantation and methods for enhancing embryonic development by genetic alteration of donor cells or by tissue culture conditions

ABSTRACT

An improved method of nuclear transfer involving the transplantation of differentiated donor cell nuclei into enucleated oocytes of a species different from the donor cell is provided. The resultant nuclear transfer units are useful for the production of isogenic embryonic stem cells, in particular human isogenic embryonic or stem cells. These embryonic or stem-like cells are useful for producing desired differentiated cells and for introduction, removal or modification, of desired genes, e.g., at specific sites of the genome of such cells by homologous recombination. These cells, which may contain a heterologous gene, are especially useful in cell transplantation therapies and for in vitro study of cell differentiation. Also, methods for improving nuclear transfer efficiency by genetically altering donor cells to inhibit apoptosis, select for a specific cell cycle and/or enhance embryonic growth and development are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of application Ser.No. 09/467,076 filed Dec. 20, 1999, which is a continuation-in-part ofapplication Ser. No. 09/395,368, filed Sep. 14, 1999, which is acontinuation-in-part of application Ser. No. 09/260,468, filed Mar. 2,1999, which is a continuation-in-part of application Ser. No.09/032,945, filed Mar. 2, 1998, which is a continuation-in-part ofapplication Ser. No. 08/699,040, filed Aug. 19, 1996, each of which isincorporated by reference in its entirety herein. application Ser. No.09/467,076 filed Dec. 20, 1999, also claims priority under 35 U.S.C. §119 to PCT/US99/04608, filed on Mar. 2, 1999, which is incorporated byreference in its entirety herein. This application is also acontinuation-in-part of application Ser. No. 09/685,061, filed Oct. 6,2000, which is a continuation-in-part of application Ser. No.09/260,468, filed Mar. 2, 1999, and which is incorporated by referencein its entirety herein. This application is also a continuation-in-partof application Ser. No. 09/874,040, filed Jun. 6, 2001, which is acontinuation-in-part of application Ser. No. 08/699,040, filed Aug.19,1996, and which is incorporated by reference in its entirety herein.This application is also a continuation-in-part of application Ser. No.09/809,018, filed Mar. 16, 2001, which is a continuation-in-part ofapplication Ser. No. 09/032,945, filed Mar. 2, 1998, and which isincorporated by reference in its entirety herein. This applicationclaims priority to provisional application 60/342,358 filed Dec. 27,2001, and to provisional application 60/357,848, filed Feb. 21, 2002,each of which is incorporated by reference in its entirety herein.

FIELD OF THE INVENTION

[0002] The present invention generally relates to the production ofembryonic or stem-like cells by the transplantation of cell nucleiderived from animal or human cells into enucleated animal oocytes of aspecies different from the donor nuclei. The present invention morespecifically relates to the production of primate or human embryonic orstem-like cells by transplantation of the nucleus of a primate or humancell into an enucleated animal oocyte, e.g., a primate or ungulateoocyte. In a preferred embodiment the donor cell or nuclei will be humanor non-human primate and the recipient cell will be an oocyte from aLagomorph, e.g., rabbit, hare or a bovine oocyte.

[0003] The present invention further relates to the use of the resultantembryonic or stem-like cells, preferably primate or human embryonic orstem-like cells for therapy, for diagnostic applications, for theproduction of differentiated cells which may also be used for therapy ordiagnosis, and for the production of transgenic embryonic or transgenicdifferentiated cells, cell lines, tissues and organs. Also, theembryonic or stem-like cells obtained according to the present inventionmay themselves be used as nuclear donors in nuclear transplantation ornuclear transfer methods for the production of chimeras or clones,preferably transgenic cloned or chimeric animals.

BACKGROUND OF THE INVENTION

[0004] Methods for deriving embryonic stem (ES) cell lines in vitro fromearly pre-implantation mouse embryos are well known. (See, e.g., Evanset al., Nature, 29:154-156 (1981); Martin, Proc. Natl. Acad. Sci., USA,78:7634-7638 (1981)). ES cells can be passaged in an undifferentiatedstate, provided that a feeder layer of fibroblast cells (Evans et al.,Id.) or a differentiation inhibiting source (Smith et al., Dev. Biol.,121:1-9 (1987)) is present.

[0005] ES cells have been previously reported to possess numerousapplications. For example, it has been reported that ES cells can beused as an in vitro model for differentiation, especially for the studyof genes that are involved in the regulation of early development. MouseES cells can give rise to germline chimeras when introduced intopre-implantation mouse embryos, thus demonstrating their pluripotency(Bradley et al., Nature, 309:255-256 (1984)).

[0006] In view of their ability to transfer their genome to the nextgeneration, ES cells have potential utility for germlne manipulation oflivestock animals by using ES cells with or without a desired geneticmodification. Moreover, in the case of livestock animals, e.g.,ungulates, nuclei from like pre-implantation livestock embryos supportthe development of enucleated oocytes to term (Smith et al., Biol.Reprod., 40:1027-1035 (1989); and Keefer et al., Biol. Reprod.,50:935-939 (1994)). This is in contrast to nuclei from mouse embryos,which beyond the eight-cell stage after transfer reportedly do notsupport the development of enucleated oocytes (Cheong et al, Biol.Reprod., 48:958 (1993)). Therefore, ES cells from livestock animals arehighly desirable because they may provide a potential source oftotipotent donor nuclei, genetically manipulated or otherwise, fornuclear transfer procedures.

[0007] Some research groups have reported the isolation of purportedlypluripotent embryonic cell lines. For example, Notarianni et al., J.Reprod. Fert. Suppl., 43:255-260 (1991), report the establishment ofpurportedly stable, pluripotent cell lines from pig and sheepblastocysts which exhibit some morphological and growth characteristicssimilar to that of cells in primary cultures of inner cell massesisolated immunosurgically from sheep blastocysts. (Id.) Also, Notarianniet al., J. Reprod. Fert. Suppl., 41:51-56 (1990) discloses maintenanceand differentiation in culture of putative pluripotential embryonic celllines from pig blastocysts. Further, Gerfen et al., Anim. Biotech,6(1):1-14 (1995) disclose the isolation of embryonic cell lines fromporcine blastocysts. These cells are stably maintained in mouseembryonic fibroblast feeder layers without the use of conditionedmedium. These cells reportedly differentiate into several different celltypes during culture (Gerfen et al., Id.).

[0008] Further, Saito et al., Roux's Arch. Dev. Biol., 201:134-141(1992) report bovine embryonic stem cell-like cell lines cultured whichsurvived passages for three, but were lost after the fourth passage.Still further, Handyside et al., Roux's Arch. Dev. Biol., 196:185-190(1987) disclose culturing of immunosurgically isolated inner cell massesof sheep embryos under conditions which allow for the isolation of mouseES cell lines derived from mouse ICMs. Handyside et al. (1987) (Id.),report that under such conditions, the sheep ICMs attach, spread, anddevelop areas of both ES cell-like and endoderm-like cells, but thatafter prolonged culture only endoderm-like cells are evident. (Id.)

[0009] Recently, Cherny et al., Theriogenology, 41:175 (1994) reportedpurportedly pluripotent bovine primordial germ cell-derived cell linesmaintained in long-term culture. These cells, after approximately sevendays in culture, produced ES-like colonies that stain positive foralkaline phosphatase (AP), exhibited the ability to form embryoidbodies, and spontaneously differentiated into at least two differentcell types. These cells also reportedly expressed mRNA for thetranscription factors OCT4, OCT6 and HES1, a pattern of homeobox geneswhich is believed to be expressed by ES cells exclusively.

[0010] Also recently, Campbell et al., Nature, 380:64-68 (1996) reportedthe production of live lambs following nuclear transfer of culturedembryonic disc (ED) cells from day nine ovine embryos cultured underconditions which promote the isolation of ES cell lines in the mouse.The authors concluded based on their results that ED cells from day nineovine embryos are totipotent by nuclear transfer and that totipotency ismaintained in culture.

[0011] Van Stekelenburg-Hamers et al., Mol. Reprod. Dev., 40:444454(1995), reported the isolation and characterization of purportedlypermanent cell lines from inner cell mass cells of bovine blastocysts.The authors isolated and cultured ICMs from 8 or 9 day bovineblastocysts under different conditions to determine which feeder cellsand culture media are most efficient in supporting the attachment andoutgrowth of bovine ICM cells. They concluded based on their resultsthat the attachment and outgrowth of cultured ICM cells is enhanced bythe use of STO (mouse fibroblast) feeder cells instead of bovine uterusepithelial cells) and by the use of charcoal-stripped serum (rather thannormal serum) to supplement the culture medium. Van Stekelenburg et alreported, however, that their cell lines resembled epithelial cells morethan pluripotent ICM cells. (Id.)

[0012] Still further, Smith et al., WO 94/24274, published Oct. 27,1994, Evans et al, WO 90/03432, published Apr. 5, 1990, and Wheeler etal, WO 94/26889, published Nov. 24, 1994, report the isolation,selection and propagation of animal stem cells which purportedly may beused to obtain transgenic animals. Also, Evans et al., WO 90/03432,published on Apr. 5, 1990, reported the derivation of purportedlypluripotent embryonic stem cells derived from porcine and bovine specieswhich assertedly are useful for the production of transgenic animals.Further, Wheeler et al, WO 94/26884, published Nov. 24, 1994, disclosedembryonic stem cells which are assertedly useful for the manufacture ofchimeric and transgenic ungulates. Thus, based on the foregoing, it isevident that many groups have attempted to produce ES cell lines, e.g.,because of their potential application in the production of cloned ortransgenic embryos and in nuclear transplantation.

[0013] The use of ungulate ICM cells for nuclear transplantation hasalso been reported. For example, Collas et al., Mol. Reprod. Dev.,38:264-267 (1994) disclose nuclear transplantation of bovine ICMs bymicroinjection of the lysed donor cells into enucleated mature oocytes.The reference disclosed culturing of embryos in vitro for seven days toproduce fifteen blastocysts which, upon transferral into bovinerecipients, resulted in four pregnancies and two births. Also, Keefer etal., Biol. Reprod., 50:935-939 (1994), disclose the use of bovine ICMcells as donor nuclei in nuclear transfer procedures, to produceblastocysts which, upon transplantation into bovine recipients, resultedin several live offspring. Further, Sims et al., Proc. Natl. Acad. Sci.,USA, 90:6143-6147 (1993), disclosed the production of calves by transferof nuclei from short-term in vitro cultured bovine ICM cells intoenucleated mature oocytes.

[0014] Also, the production of live lambs following nuclear transfer ofcultured embryonic disc cells has been reported (Campbell et al.,Nature, 380:64-68 (1996)). Still further, the use of bovine pluripotentembryonic cells in nuclear transfer and the production of chimericfetuses have also been reported (Stice et al., Biol. Reprod., 54:100-110(1996)); Collas et al, Mol. Reprod. Dev., 38:264-267 (1994).

[0015] Further, there have been previous attempts to produce crossspecies NT units (Wolfe et al., Theriogenology, 33:350 (1990).Specifically, bovine embryonic cells were fused with bison oocytes toproduce some cross species NT units possibly having an inner cell mass.However, embryonic cells, not adult cells were used, as donor nuclei inthe nuclear transfer procedure. The dogma has been that embryonic cellsare more easily reprogrammed than adult cells. This dates back toearlier NT studies in the frog (review by DiBerardino, Differentiation,17:17-30 (1980)). Also, this study involved very phylogeneticallysimilar animals (cattle nuclei and bison oocytes). By contrast,previously when more diverse species were fused during NT (cattle nucleiinto hamster oocytes), no inner cell mass structures were obtained.Further, it has never been previously reported that the inner cell masscells from NT units could be used to form an ES cell-like colony thatcould be propagated.

[0016] Also, Collas et al (Id.), taught the use of granulosa cells(adult somatic cells) to produce bovine nucleartransfer embryos.However, unlike the present invention, these experiments did not involvecross-species nuclear transfer. Also, unlike the present inventionES-like cell colonies were not obtained.

[0017] Recently, U.S. Pat. No. 5,843,780, issued to James A. Thomson onDec. 1, 1998, assigned to the Wisconsin Alumni Research Foundation,purports to disclose a purified preparation of primate embryonic stemcells that are (i) capable of proliferation in an in vitro culture forover one year; (ii) maintain a karyotype in which all chromosomescharacteristic of the primate species are present and not noticeablyaltered through prolonged culture; (iii) maintains the potential todifferentiate into derivatives of endoderm, mesoderm and ectodermtissues throughout culture; and (iv) will not differentiate whencultured on a fibroblast feeder layer. These cells were reportedlynegative for the SSEA-1 marker, positive for the SEA-3 marker, positivefor the SSEA-4 marker, express alkaline phosphatase activity, arepluripotent, and have karyotypes which include the presence of all thechromosomes characteristic of the primate species and in which none ofthe chromosomes are altered. Further, these cells are respectfullypositive for the TRA-1-60, and TRA-1-81 markers. The cells purportedlydifferentiate into endoderm, mesoderm and ectoderm cells when injectedinto a SCID mouse. Also, purported embryonic stem cell lines derivedfrom human or primate blastocysts are discussed in Thomson et al.,Science 282:1145-1147 and Proc. Natl. Acad. Sci., USA 92:7844-7848(1995).

[0018] Thus, Thomson discloses what purportedly are non-human primateand human embryonic or stem-like cells and methods for their production.However, there still exists a significant need for methods for producinghuman embryonic or stem-like cells that are autologous to an intendedtransplant recipient given their significant therapeutic and diagnosticpotential.

[0019] In this regard, numerous human diseases have been identifiedwhich may be treated by cell transplantation. For example, Parkinson'sdisease is caused by degeneration of dopaminergic neurons in thesubstantial nigra. Standard treatment for Parkinson's involvesadministration of L-DOPA, which temporarily ameliorates the loss ofdopamine, but causes severe side effects and ultimately does not reversethe progress of the disease. A different approach to treatingParkinson's, which promises to have broad applicability to treatment ofmany brain diseases and central nervous system injury, involvestransplantation of cells or tissues from fetal or neonatal animals intothe adult brain. Fetal neurons from a variety of brain regions can beincorporated into the adult brain. Such grafts have been shown toalleviate experimentally induced behavioral deficits, including complexcognitive functions, in laboratory animals. Initial test results fromhuman clinical trials have also been promising. However, supplies ofhuman fetal cells or tissue obtained from miscarriages is very limited.Moreover, obtaining cells or tissues from aborted fetuses is highlycontroversial.

[0020] There is currently no available procedure for producing“fetal-like” cells from the patient. Further, allograft tissue is notreadily available and both allograft and xenograft tissue are subject tograft rejection. Moreover, in some cases, it would be beneficial to makegenetic modifications in cells or tissues before transplantation.However, many cells or tissues wherein such modification would bedesirable do not divide well in culture and most types of genetictransformation require rapidly dividing cells.

[0021] There is therefore a clear need in the art for a supply of humanembryonic or stem-like undifferentiated cells for use in transplants andcell and gene therapies.

OBJECTS OF THE INVENTION

[0022] It is an object of the invention to provide novel and improvedmethods for producing embryonic or stem-like cells.

[0023] It is a more specific object of the invention to provide a novelmethod for producing embryonic or stem-like cells which involvestransplantation of the nucleus of a vertebrate cell, e.g., a mammal,reptile, amphibian or avian into a suitable recipient cell, e.g., anoocyte of a different species.

[0024] It is more specific object of the invention to provide a novelmethod for producing non-human primate or human embryonic or stem-likecells which involves transplantation of the nucleus of a non-humanprimate or human cell into an a recipient cell, e.g., an animal or humanoocyte, e.g., an ungulate, human or primate enucleated oocyte or aLagomorph oocyte.

[0025] It is another object of the invention to enhance the efficacy ofcross-species nuclear transfer by incorporating mitochondrial DNAderived from the same species (preferably same donor) as the donor cellinto the oocyte of a different species that is used for nucleartransfer, before or after enucleation, or into the nuclear transfer unit(after the donor cell has been introduced). Preferably, the donor cellor nucleus will be a human or non-human primate cell or nucleus and therecipient cell will be an oocyte, e.g., or ungulate or Lagomorph.

[0026] It is still another object of the invention to enhance theefficacy of cross-species nuclear transfer by fusing an enucleatedsomatic cell (e.g., an enucleated human somatic cell) (karyoplast) withan activated or non-activated, enucleated or non-enucleated oocyte of adifferent species, e.g., bovine, or by fusion with an activated orunactivated cross-species NT unit which may be cleaved or uncleaved.

[0027] It is another object of the invention to provide a novel methodfor producing lineage-defective non-human primate or human embryonic orstem-like cells which involves transplantation of the nucleus of anon-human primate or human cell, e.g., a human adult cell into anenucleated non-human primate or human oocyte, wherein such cell has beengenetically engineered to be incapable of differentiation into aspecific cell lineage or has been modified such that the cells are“mortal”, and thereby do not give rise to a viable offspring, e.g., byengineering expression of anti-sense or ribozyme telomerase gene.

[0028] It is still another object of the invention to enhance efficiencyof nuclear transfer and specifically to enhance the development ofpre-implantation embryos produced by nuclear transfer by geneticallyengineering donor somatic cells used for nuclear transfer to provide forthe expression of genes that enhance embryonic development, e.g., genesof the MHC I family, and in particular Ped genes such as Q7 and/or Q9.

[0029] It is another object of the invention to enhance the productionof nuclear transfer embryos, e.g., cross-species nuclear transferembryos, by the introduction of transgenes before or after nucleartransfer that provide for the expression of an antisense DNA encoding acell death gene such as BAX, Apaf-1, or capsase, or a portion thereof,or demethylase.

[0030] It is yet another object of the invention to enhance theproduction of nuclear transfer embryos by IVP and more specificallynuclear transfer embryos by genetically altering the donor cell used fornuclear transfer such that it is resistant to apoptosis, e.g., byintroduction of a DNA construct that provides for the expression ofgenes that inhibit apoptosis, e.g., Bcl-2 or Bcl-2 family members and/orby the expression of antisense ribozymes specific to genes that induceapoptosis during early embryonic development.

[0031] It is still another object of the invention to improve theefficacy of nuclear transfer by improved selection of donor cells of aspecific cell cycle stage, e.g., G1 phase, by genetically engineeringdonor cells such that they express a DNA construct encoding a particularcyclin linked to a detectable marker, e.g., one that encodes avisualizable (e.g., fluorescent tag) marker protein.

[0032] It is also an object of the invention to enhance the developmentof in vitro produced embryos, by culturing such embryos in the presenceof one or more protease inhibitors, preferably one or more capsaseinhibitors, thereby inhibiting apoptosis.

[0033] It is another object of the invention to provide embryonic orstem-like cells produced by transplantation of nucleus of an animal orhuman cell into an enucleated oocyte of a different species.

[0034] It is a more specific object of the invention to provide primateor human embryonic or stem-like cells produced by transplantation of thenucleus of a primate or human cell into an enucleated animal oocyte,e.g., a human, primate or ungulate enucleated oocyte.

[0035] It is another object of the invention to use such embryonic orstem-like cells for therapy or diagnosis.

[0036] It is a, specific object of the invention to use such primate orhuman embryonic or stem-like cells for treatment or diagnosis of anydisease wherein cell, tissue or organ transplantation is therapeuticallyor diagnostically beneficial.

[0037] It is another specific object of the invention to use theembryonic or stem-like cells produced according to the invention for theproduction of differentiated cells, tissues or organs. In a preferredembodiment these cells will be obtained by fusion or implantation of ahuman cell or nucleus into a recipient oocyte, e.g., an ungulate (bovineetc.) or a lagomorph, e.g., a rabbit, hare or pika.

[0038] It is a more specific object of the invention to use the primateor human embryonic or stem-like cells produced according to theinvention for the production of differentiated human cells, tissues ororgans.

[0039] It is another specific object of the invention to use theembryonic or stem-like cells produced according to the invention for theproduction of genetically engineered embryonic or stem-like cells, whichcells may be used to produce genetically engineered or transgenicdifferentiated human cells, tissues or organs, e.g., having use in genetherapies.

[0040] It is another specific object of the invention to use theembryonic or stem-like cells produced according to the invention invitro, e.g. for study of cell differentiation and for assay purposes,e.g. for drug studies.

[0041] It is another object of the invention to provide improved methodsof transplantation therapy, comprising the usage of isogenic orsynegenic cells, tissues or organs produced from the embryonic orstem-like cells produced according to the invention. Such therapiesinclude by way of example treatment of diseases and injuries includingParkinson's, Huntington's, Alzheimer's, ALS, spinal cord injuries,multiple sclerosis, muscular dystrophy, diabetes, liver diseases, heartdisease, cartilage replacement, burns, vascular diseases, urinary tractdiseases, as well as for the treatment of immune defects, bone marrowtransplantation, cancer, among other diseases.

[0042] It is another object of the invention to use the transgenic orgenetically engineered embryonic or stem-like cells produced accordingto the invention for gene therapy, in particular for the treatmentand/or prevention of the diseases and injuries identified, supra.

[0043] It is another object of the invention to use the embryonic orstem-like cells produced according to the invention or transgenic orgenetically engineered embryonic or stem-like cells produced accordingto the invention as nuclear donors for nuclear transplantation.

[0044] It is still another object of the invention to use geneticallyengineered ES cells produced according to the invention for theproduction of transgenic animals, e.g., non-human primates, rodents,ungulates, etc. Such transgenic animals can be used to produce, e.g.,animal models for study of human diseases, or for the production ofdesired polypeptides, e.g., therapeutics or nutripharmaceuticals.

[0045] With the foregoing and other objects, advantages and features ofthe invention that will become hereinafter apparent, the nature of theinvention may be more clearly understood by reference to the followingdetailed description of the preferred embodiments of the invention andto the appended claims.

BRIEFS DESCRIPTION OF THE FIGURES

[0046]FIG. 1 is a photograph of a nuclear transfer (NT) unit produced bytransfer of an adult human cell into an enucleated bovine oocyte.

[0047] FIGS. 2 to 5 are photographs of embryonic stem-like cells derivedfrom a NT unit such as is depicted in FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention provides a novel method for producingembryonic or stem-like cells, and more specifically non-human primate orhuman embryonic or stem-like cells by nuclear transfer or nucleartransplantation. In the subject application, nuclear transfer or nucleartransplantation or NT are used interchangeably.

[0049] As discussed supra, the isolation of actual embryonic orstem-like cells by nuclear transfer or nuclear transplantation has neverbeen reported. Rather, previous reported isolation of ES-like cells hasbeen from fertilized embryos. Also, successful nuclear transferinvolving cells or DNA of genetically dissimilar species, or morespecifically adult cells or DNA of one species (e.g., human) and oocytesof another non-related species has never been reported. Rather, whileembryos produced by fusion of cells of closely related species, has beenreported, e.g., bovine-goat and bovine-bison, they did not produce EScells. (Wolfe et al, Theriogenology, 33(1):350 (1990).) Also, there hasnever been reported a method for producing primate or human ES cellsderived from a non-fetal tissue source. Rather, the limited human fetalcells and tissues which are currently available must be obtained orderived from spontaneous abortion tissues and from aborted fetuses.

[0050] Also, prior to the present invention, no one obtained embryonicor stem-like cells by cross-species nuclear transplantation.

[0051] Quite unexpectedly, the present inventors discovered that humanembryonic or stem-like cells and cell colonies may be obtained bytransplantation of the nucleus of a human cell, e.g., an adultdifferentiated human cell, into an enucleated animal oocyte, which isused to produce nuclear transfer (NT) units, the cells of which uponculturing give rise to human embryonic or stem-like cells and cellcolonies. This result is highly surprising because it is the firstdemonstration of effective cross-species nuclear transplantationinvolving the introduction of a differentiated donor cell or nucleusinto an enucleated oocyte of a genetically dissimilar species, e.g., thetransplantation of cell nuclei from a differentiated animal or humancell, e.g., adult cell, into the enucleated egg of a different animalspecies, to produce nuclear transfer units containing cells which whencultured under appropriate conditions give rise to embryonic orstem-like cells and cell colonies. In preferred embodiments, the donorcell or nucleus and recipient cell (oocyte) will be of different orderorganism. For example, in a preferred embodiment the donor cell ornucleus is human or non-human primate and the recipient cell (oocyte) isan ungulate or Lagomorpha oocyte, preferably a rabbit or hare. Examples,thereof, including domesticated rabbits, jack rabbits, hares,cottontails, snowshoe and others. This order includes animals of thegenera lepus, sylvilagus and oryctolugus.

[0052] In a preferred embodiment nuclear transfer units producedaccording to the invention will be allowed to develop into blastocystsor morula stage embryos and these blastocysts, morula stage embryos orportions thereof, e.g., the inner cell mass will be induced todifferentiate into desired cell lineages by contacting with differentgrowth factors, hormones and other substituents that induce celldifferentiate. Alternatively, differentiation can be effected in viro byimplantation into a suitable surrogate animal.

[0053] Preferably, the NT units used to produce ES-like cells will becultured to a size of at least 2 to 400 cells, preferably 4 to 128cells, and most preferably to a size of at least about 50 cells.

[0054] In the present invention, embryonic or stem-like cells refer tocells produced according to the present invention. The presentapplication refers to such cells as stem-like cells rather than stemcells because of the manner in which they are typically produced, i.e.,by cross-species nuclear transfer. While these cells are expected topossess similar differentiation capacity as normal stem cells they maypossess some insignificant differences because of the manner they areproduced. For example, these stem-like cells may possess themitochondria of the oocytes used for nuclear transfer, and thus notbehave identically to conventional embryonic stem cells.

[0055] The present discovery was made based on the observation thatnuclear transplantation of the nucleus of an adult human cell,specifically a human epithelial cell obtained from the oral cavity of ahuman donor, when transferred into an enucleated bovine oocyte, resultedin the formation of nuclear transfer units, the cells of which uponculturing gave rise to human stem-like or embryonic cells and humanembryonic or stem-like cell colonies. This result has recently beenreproduced by transplantation of keratinocytes from an adult human intoan enucleated bovine oocyte with the successful production of ablastocyst and ES cell line. Based thereon, it is hypothesized by thepresent inventors that bovine oocytes and human oocytes, and likelymammals in general must undergo maturation processes during embryonicdevelopment which are sufficiently similar or conserved so as to permitthe bovine oocyte to function as an effective substitute or surrogatefor a human oocyte. Apparently, oocytes in general comprise factors,likely proteinaceous or nucleic acid in nature, that induce embryonicdevelopment under appropriate conditions, and these functions that arethe same or very similar in different species. These factors maycomprise material RNAs and/or telomerase.

[0056] Based on the fact that human cell nuclei can be effectivelytransplanted into bovine oocytes, it is reasonable to expect that humancells may be transplanted into oocytes of other non-related species,e.g., other ungulates as well as other animals. In particular, otherungulate oocytes should be suitable, e.g. pigs, sheep, horses, goats,etc. Also, oocytes from other sources should be suitable, e.g. oocytesderived from other primates, amphibians, rodents, rabbits, guinea pigs,etc. Further, using similar methods, it should be possible to transferhuman cells or cell nuclei into human oocytes and use the resultantblastocysts to produce human ES cells.

[0057] In fact, as disclosed in an exemplified protocol infra in theexamples, the present inventor have produced blastocysts by fusion of ahuman donor cell and a rabbit oocyte which has been enucleated to removeits endogenous nucleus.

[0058] Therefore, in its broadest embodiment, the present inventioninvolves the transplantation of human cell nucleus or animal or humancell into an oocyte (preferably enucleated) of an animal speciesdifferent from the donor nuclei, by injection or fusion, to produce anNT unit containing cells which may be used to obtain embryonic orstem-like cells and/or cell cultures. Enucleation (removal of endogenousoocyte nucleus) may be effected before or after nuclear transfer. Forexample, the invention may involve the transplantation of an ungulatecell nucleus or ungulate cell into an enucleated oocyte of anotherspecies, e.g., another ungulate or non-ungulate, by injection or fusion,which cells and/or nuclei are combined to produce NT units and which arecultured under conditions suitable to obtain multicellular NT units,preferably comprising at least about 2 to 400 cells, more preferably 4to 128 cells, and most preferably at least about 50 cells. The cells ofsuch NT units may be used to produce embryonic or stem-like cells orcell colonies upon culturing.

[0059] However, the preferred embodiment of the invention comprises theproduction of non-human primate or human embryonic or stem-like cells bytransplantation of the nucleus of a donor human cell or a human cellinto an enucleated human, primate, or non-primate animal oocyte, e.g.,an ungulate oocyte, and in a preferred embodiment a bovine enucleatedoocyte or a lagomorpha oocyte, e.g., rabbit, hare or pika.

[0060] In general, the embryonic or stem-like cells will be produced bya nuclear transfer process comprising the following steps:

[0061] (i) obtaining desired human or animal cells to be used as asource of donor nuclei (which may be genetically altered);

[0062] (ii) obtaining oocytes from a suitable source, e.g. a mammal andmost preferably a primate or an ungulate source, e.g. bovine,

[0063] (iii) enucleating said oocytes by removal of endogenous nucleus;

[0064] (iv) transferring the human or animal cell or nucleus into theenucleated oocyte of an animal species different than the donor cell ornuclei, e.g., by fusion or injection, wherein steps (iii) and (iv) maybe effected in either order;

[0065] (v) culturing the resultant NT product or NT unit to producemultiple cell structures (embryoid structures having a discernible innercell mass); and

[0066] (vi) culturing cells obtained from said embryos to obtainembryonic or stem-like cells and stem-like cell colonies.

[0067] Nuclear transfer techniques or nuclear transplantation techniquesare known in the literature and are described in many of the referencescited in the Background of the Invention. See, in particular, Campbellet al, Theriogenology, 43:181 (1995); Collas et al, Mol. Report Dev.,38:264-267 (1994); Keefer et al, Biol. Reprod., 50:935-939 (1994); Simset al, Proc. Natl. Acad. Sci., USA, 90:6143-6147 (1993); WO 94/26884; WO94/24274, and WO 90/03432, which are incorporated by reference in theirentirety herein. Also, U.S. Pat. Nos. 4,944,384 and 5,057,420 describeprocedures for bovine nuclear transplantation. See, also Cibelli et al,Science, Vol. 280:1256-1258 (1998).

[0068] Human or animal cells, preferably mammalian cells, may beobtained and cultured by well known methods. Human and animal cellsuseful in the present invention include, by way of example, epithelial,neural cells, epidermal cells, keratinocytes, hematopoietic cells,melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), otherimmune cells, erythrocytes, macrophages, melanocytes, monocytes,mononuclear cells, fibroblasts, cardiac muscle cells, cumulus cells andother muscle cells, etc. Moreover, the human cells used for nucleartransfer may be obtained from different organs, e.g., skin, lung,pancreas, liver, stomach, intestine, heart, reproductive organs,bladder, kidney, urethra and other urinary organs, etc. These are justexamples of suitable donor cells. Suitable donor cells, i.e., cellsuseful in the subject invention, may be obtained from any cell or organof the body. This includes all somatic or germ cells e.g., primordialgerm cells, sperm cells. Preferably, the donor cells or nucleus canactively dividing, i.e., non-quiescent, cells as this has been reportedto enhance cloning efficacy. Such cells include those in the G1, G2 S orM cell phase. Alternatively, quiescent cells may be used. Alsopreferably, such donor cells will be in the G1 cell cycle.

[0069] The resultant blastocysts may be used to obtain embryonic stemcell lines according to the culturing methods reported by Thomson etal., Science, 282:1145-1147 (1998) and Thomson et al., Proc. Natl. Acad.Sci., USA, 92:7544-7848 (1995), incorporated by reference in theirentirety herein.

[0070] In one Example disclosed infra, the cells used as donors fornuclear transfer were epithelial cells derived from the oral cavity of ahuman donor and adult human keratinocytes. However, as discussed, thedisclosed method is applicable to other human cells or nuclei. Moreover,the cell nuclei may be obtained from both human somatic and germ cells.In the second example, the donor cell isa human cell, somatic cell andthe recipient cell is an enucleated rabbit oocyte. The fact that bothrabbit oocytes and bovine oocytes upon fusion with a human cell yieldnuclear transfer units that clear and give rise to which appears to be acell cloning an exhibiting ES-like appearance supports Applicants beliefthat the application of a variety of different species, very distinctfrom primate can be used to reprogram the nuclei of a human cell. It isanticipated especially that other mammalian cells will be suitable,under appropriate culture and activation conditions.

[0071] It is also possible to arrest donor cells at mitosis beforenuclear transfer, using a suitable technique known in the art. Methodsfor stopping the cell cycle at various stages have been thoroughlyreviewed in U.S. Pat. No. 5,262,409, which is herein incorporated byreference. In particular, while cycloheximide has been reported to havean inhibitory effect on mitosis (Bowen and Wilson (1955) J. Heredity,45:3-9), it may also be employed for improved activation of maturebovine follicular oocytes when combined with electric pulse treatment(Yang et al. (1992) Biol. Reprod., 42 (Suppl. 1): 117).

[0072] Zygote gene activation is associated with hyperacetylation ofHistone H4. Trichostatin-A has been shown to inhibit histone deacetylasein a reversible manner (Adenot et al. Differential H4 acetylation ofpaternal and maternal chromatin precedes DNA replication anddifferential transcriptional activity in pronuclei of 1-cell mouseembryos. Development (November 1997) 124(22): 4615-4625; Yoshida et al.Trichostatin A and trapoxin: novel chemical probes for the role ofhistone acetylation in chromatin structure and function. Bioessays (May,1995) 17(5): 423430), as have other compounds. For instance, butyrate isalso believed to cause hyper-acetylations of histones by inhibitinghistone deacetylase. Generally, butyrate appears to modify geneexpression and in almost all cases its addition to cells in cultureappears to arrest cell growth. Use of butyrate in this regard isdescribed in U.S. Pat. No. 5,681,718, which is herein incorporated byreference. Thus, donor cells may be exposed to Trichostatin-A or anotherappropriate deacetylase inhibitor prior to fusion, or such a compoundmay be added to the culture media prior to genome activation.

[0073] Additionally, demethylation of DNA is thought to be a requirementfor proper access of transcription factors to DNA regulatory sequences.Global demethylation of DNA from the eight-cell stage to the blastocyststage in pre-implantation embryos has previously been described (Steinet al., Mol. Reprod. & Dev., 47(4): 421-429). Also, Jaenisch et al.(1997) have reported that 5-azacytidine can be used to reduce the levelof DNA methylation in cells, potentially leading to increased access oftranscription factors to DNA regulatory sequences. Accordingly, donorcells may be exposed to 5-azacytidine (5-Aza) previous to fusion, or5-Aza may be added to the culture medium from the 8 cell stage toblastocyst. Alternatively, other known methods for effecting DNAdemethylation may be used.

[0074] The oocytes used for nuclear transfer may be obtained fromanimals including mammals, avians, reptiles and amphibians. Suitablemammalian sources for oocytes include sheep, bovines, ovines, pigs,horses, rabbits, goats, guinea pigs, mice, hamsters, rats, primates,humans, etc. In the preferred embodiments, the oocytes will be obtainedfrom primates, lagomorphs or ungulates, e.g., a bovine or rabbits.

[0075] Methods for isolation of oocytes are well known in the art.Essentially, this will comprise isolating oocytes from the ovaries orreproductive tract of a mammal or amphibian, e.g., a bovine. A readilyavailable source of bovine oocytes is slaughterhouse materials. Rabbitoocytes are also readily available. As noted, enucleation may beeffected before or after nuclear or cell transplantation.

[0076] For the successful use of techniques such as genetic engineering,nuclear transfer and cloning, oocytes are preferably matured in vitrobefore these cells may be used as recipient cells for nuclear transfer,and before they can be fertilized by the sperm cell to develop into anembryo. This process generally requires collecting immature (prophase I)oocytes from animal ovaries, e.g., bovine ovaries obtained at aslaughterhouse and maturing the oocytes in a maturation medium prior tofertilization or enucleation until the oocyte attains the metaphase IIstage, which in the case of bovine oocytes generally occurs about 18-24hours post-aspiration. For purposes of the present invention, thisperiod of time is known as the “maturation period.” As used herein forcalculation of time periods, “aspiration” refers to aspiration of theimmature oocyte from ovarian follicles.

[0077] Additionally, metaphase II stage oocytes, which have been maturedin vivo have been successfully used in nuclear transfer techniques.Essentially, mature metaphase II oocytes are collected surgically fromeither non-superovulated or superovulated cows or heifers 35 to 48 hourspast the onset of estrus or past the injection of human chorionicgonadotropin (hCG) or similar hormone. Alternatively, metaphase Ioocytes may be utilized.

[0078] The stage of maturation of the oocyte at enucleation and nucleartransfer has been reported to be significant to the success of NTmethods. (See e.g., Prather et al., Differentiation, 48, 1-8, 1991). Ingeneral, previous successful mammalian embryo cloning practices usedmetaphase 11 stage oocyte as the recipient oocyte because at this stageit is believed that the oocyte can be or is sufficiently “activated” totreat the introduced nucleus as it does a fertilizing sperm. In domesticanimals, and especially cattle, the oocyte activation period generallyranges from about 16-52 hours, preferably about 28-42 hourspost-aspiration.

[0079] For example, immature oocytes may be washed in HEPES bufferedhamster embryo culture medium (HECM) as described in Seshagine et al.,Biol. Reprod., 40, 544-606, 1989, and then placed into drops ofmaturation medium consisting of 50 microliters of tissue culture medium(TCM) 199 containing 10% fetal calf serum which contains appropriategonadotropins such as luteinizing hormone (LH) and follicle stimulatinghormone (FSH), and estradiol under a layer of lightweight paraffin orsilicon at 39° C.

[0080] After a fixed time maturation period, which typically will rangefrom about 10 to 40 hours, and preferably about 16-18 hours, the oocyteswill typically be enucleated. Prior to enucleation the oocytes willpreferably be removed and placed in HECM containing I milligram permilliliter of hyaluronidase prior to removal of cumulus cells. This maybe effected by repeated pipetting through very fine bore pipettes or byvortexing briefly. The stripped oocytes are then screened for polarbodies, and the selected metaphase 11 oocytes, as determined by thepresence of polar bodies, are then used for nuclear transfer.Enucleation follows. As noted above, enucleation may be effected beforeor after introduction of donor cell or nucleus because the donor nucleusis readily discernible from endogenous nucleus.

[0081] Enucleation may be effected by known methods, such as describedin U.S. Pat. No. 4,994,384 which is incorporated by reference herein.For example, metaphase II oocytes are either placed in HECM, optionallycontaining 7.5 micrograms per milliliter cytochalasin B, for immediateenucleation, or may be placed in a suitable medium, for example CR1aa,plus 10% estrus cow serum, and then enucleated later, preferably notmore than 24 hours later, and more preferably 16-18 hours later.

[0082] Enucleation may be accomplished microsurgically using amicropipette to remove the polar body and the adjacent cytoplasm. Theoocytes may then be screened to identify those of which have beensuccessfully enucleated. This screening may be effected by staining theoocytes with 1 microgram per milliliter 33342 Hoechst dye in HECM, andthen viewing the oocytes under ultraviolet irradiation for less than 10seconds. The oocytes that have been successfully enucleated can then beplaced in a suitable culture medium.

[0083] In the present invention, the recipient oocytes will typically beenucleated at a time ranging from about 10 hours to about 40 hours afterthe initiation of in vitro maturation, more preferably from about 16hours to about 24 hours after initiation of in vitro maturation, andmost preferably about 16-18 hours after initiation of in vitromaturation. Enucleation may be effected before, simultaneous or afternuclear transfer. Also, enucleation may be effected before, after orsimultaneous to activation.

[0084] A single animal or human cell or nucleus derived therefrom whichis typically heterologous to the enucleated oocyte will then betransferred into the perivitelline space of the oocyte, typicallyenucleated, used to produce the NT unit. However, removal of endogenousnucleus may alternatively be effected after nuclear transfer. The animalor human cell or nucleus and the enucleated oocyte will be used toproduce NT units according to methods known in the art. For example, thecells may be fused by electrofusion. Electrofusion is accomplished byproviding a pulse of electricity that is sufficient to cause a transientbreak down of the plasma membrane. This breakdown of the plasma membraneis very short because the membrane reforms rapidly. Essentially, if twoadjacent membranes are induced to break down, upon reformation the lipidbilayers intermingle and small channels will open between the two cells.Due to the thermodynamic instability of such a small opening, itenlarges until the two cells become one. Reference is made to U.S. Pat.No. 4,997,384, by Prather et al., (incorporated by reference in itsentirety herein) for a further discussion of this process. A variety ofelectrofusion media can be used including e.g., sucrose, mannitol,sorbitol and phosphate buffered solution. Fusion can also beaccomplished using Sendai virus as a fusogenic agent (Graham, WisterInot. Symp. Monogr., Sep. 19, 1969).

[0085] Also, in some cases (e.g. with small donor nuclei) it may bepreferable to inject the nucleus directly into the oocyte rather thanusing electroporation fusion. Such techniques are disclosed in Collasand Barnes, Mol. Reprod. Dev., 38:264-267 (1994), and incorporated byreference in its entirety herein.

[0086] Preferably, the human or animal cell and oocyte are electrofusedin a 500 μm chamber by application of an electrical pulse of 90-120V forabout 15 μsec, about 24 hours after initiation of oocyte maturation.After fusion, the resultant fused NT units are preferably placed in asuitable medium until activation, e.g., one identified infra. Typicallyactivation will be effected shortly thereafter, typically less than 24hours later, and preferably about 4-9 hours later. However, it is alsopossible to activate the recipient oocyte before or proximate(simultaneous) to nuclear transfer, which may or may not be enucleated.For example, activation may be effected from about twelve hours prior tonuclear transfer to about twenty-four hours after nuclear transfer. Moretypically, activation is effected simultaneous or shortly after nucleartransfer, e.g., about four to nine hours later.

[0087] The NT unit may be activated by known methods. Such methodsinclude, e.g., culturing the NT unit at sub-physiological temperature,in essence by applying a cold, or actually cool temperature shock to theNT unit. This may be most conveniently done by culturing the NT unit atroom temperature, which is cold relative to the physiologicaltemperature conditions to which embryos are normally exposed.

[0088] Alternatively, activation may be achieved by application of knownactivation agents. For example, penetration of oocytes by sperm duringfertilization has been shown to activate prefusion oocytes to yieldgreater numbers of viable pregnancies and multiple genetically identicalcalves after nuclear transfer. Also, treatments such as electrical andchemical shock or cycloheximide treatment may also be used to activateNT embryos after fusion. Suitable oocyte activation methods are thesubject of U.S. Pat. No. 5,496,720, to Susko-Parrish et al., which isherein incorporated by reference.

[0089] For example, oocyte activation may be effected by simultaneouslyor sequentially:

[0090] (i) increasing levels of divalent cations in the oocyte, and

[0091] (ii) reducing phosphorylation of cellular proteins in the oocyte.

[0092] This will generally be effected by introducing divalent cationsinto the oocyte cytoplasm, e.g., magnesium, strontium, barium orcalcium, e.g., in the form of an ionophore. Other methods of increasingdivalent cation levels include the use of electric shock, treatment withethanol and treatment with caged chelators.

[0093] Phosphorylation may be reduced by known methods, e.g., by theaddition of kinase inhibitors, e.g., serine-threonine kinase inhibitors,such as 6-dimethylamino-purine, staurosporine, 2-aminopurine, andsphingosine.

[0094] Alternatively, phosphorylation of cellular proteins may beinhibited by introduction of a phosphatase into the oocyte, e.g.,phosphatase 2A and phosphatase 2B.

[0095] Specific examples of activation methods are listed below.

[0096] 1. Activation by lonomycin and DMAP

[0097] 1—Place oocytes in lonomycin (5 μM) with 2 mM of DMAP for 4minutes;

[0098] 2—Move the oocytes into culture media with 2 mM of DMAP for 4hours;

[0099] 3—Rinse four times and place in culture.

[0100] 2. Activation by lonomycin DMAP and Roscovitin

[0101] 1—Place oocytes in lonomycin (5 μM) with 2 mM of DMAP for fourminutes;

[0102] 2—Move the oocytes into culture media with 2 mM of DMAP and 200microM of Roscovitin for three hours;

[0103] 3—Rinse four times and place in culture.

[0104] 3. Activation by exposure to lonomycin followed by cytochalasinand cycloheximide.

[0105] 1—Place oocytes in lonomycin (5 microM) for four minutes;

[0106] 2—Move oocytes to culture media containing 5 μg/ml ofcytochalasin B and 5 μg/ml of cycloheximide for five hours;

[0107] 3—Rinse four times and place in culture.

[0108] 4. Activation by electrical pulses

[0109] 1—Place eggs in mannitol media containing 100 μM CaCL₂;

[0110] 2—Deliver three pulses of 1.0 kVcm⁻¹ for 20 μsec, each pulse 22minutes apart;

[0111] 3—Move oocytes to culture media containing 5 μg/ml ofcytochalasin B for three hours.

[0112] 5. Activation by exposure with ethanol followed by cytochalasinand cycloheximide

[0113] 1—Place oocytes in 7% ethanol for one minute;

[0114] 2—Move oocytes to culture media containing 5 μg/ml ofcytochalasin. B and 5 μg/ml of cycloheximide for five hours;

[0115] 3—Rinse four times and place in culture.

[0116] 6. Activation by microinjection of adenophostine

[0117] 1—Inject oocytes with 10 to 12 picoliters of a solutioncontaining 10 μM of adenophostine;

[0118] 2—Put oocytes in culture.

[0119] 7. Activation by microinjection of sperm factor

[0120] 1 —Inject oocytes with 10 to 12 picoliters of sperm factorisolated, e.g., from primates, pigs, bovine, sheep, goats, horses, mice,rats, rabbits or hamsters;

[0121] 2—Put eggs in culture.

[0122] 8. Activation by microinjection of recombinant sperm factor.

[0123] 9. Activation by Exposure to DMAP followed by Cycloheximide andCytochalasin B

[0124] Place oocytes or NT units, typically about 22 to 28 hours postmaturation in about 2 mM DMAP for about one hour, followed by incubationfor about two to twelve hours, preferably about eight hours, in 5 pg/mlof cytochalasin B and 20 μg/ml cycloheximide.

[0125] The above activation protocols are exemplary of protocols usedfor nuclear transfer-procedures, e.g., those including the use ofprimate or human donor cells or oocytes. However, the above activationprotocols may be used when either or both the donor cell and nucleus isof ungulate origin, e.g., a sheep, buffalo, horse, goat, bovine, pigand/or wherein the oocyte is of ungulate origin, e.g., sheet, pig,buffalo, horse, goat, bovine, etc., as well as for other species, e.g.,Lagomorphs such as rabbits and hares.

[0126] As noted, activation may be effected before, simultaneous, orafter nuclear transfer. In general, activation will be effected about 40hours prior to nuclear transfer and fusion to about 40 hours afternuclear transfer and fusion, more preferably about 24 hours before toabout 24 hours after nuclear transfer and fusion, and most preferablyfrom about 4 to 9 hours before nuclear transfer and fusion to about 4 to9 hours after nuclear transfer and fusion. Activation is preferablyeffected after or proximate to in vitro or in vivo maturation of theoocyte, e.g., approximately simultaneous or within about 40 hours ofmaturation, more preferably within about 24 hours of maturation.

[0127] Activated NT units may be cultured in a suitable in vitro culturemedium until the generation of embryonic or stem-like cells and cellcolonies. Culture media suitable for culturing and maturation of embryosare well known in the art. Examples of known media, which may be usedfor bovine embryo culture and maintenance, include Ham's F-10+10% fetalcalf serum (FCS), Tissue Culture Medium-199 (TCM-199)+10% fetal calfserum, Tyrodes-Albumin-Lactate-Pyruvate (TALP), Dulbecco's PhosphateBuffered Saline (PBS), Eagle's and Whitten's media. One of the mostcommon media used for the collection and maturation of oocytes isTCM-199, and 1 to 20% serum supplement including fetal calf serum,newborn serum, estrual cow serum, lamb serum or steer serum. A preferredmaintenance medium includes TCM-199 with Earl salts, 10% fetal calfserum, 0.2 Ma pyruvate and 50 μg/ml gentamicin sulphate. Any of theabove may also involve co-culture with a variety of cell types such asgranulosa cells, oviduct cells, BRL cells and uterine cells and STOcells.

[0128] In particular, human epithelial cells of the endometrium secreteleukemia inhibitory factor (LIF) during the preimplantation andimplantation period. Therefore, the addition of LIF to the culturemedium could be of importance in enhancing the in vitro development ofthe reconstructed embryos. The use of LIF for embryonic or stem-likecell cultures has been described in U.S. Pat. No. 5,712,156, which isherein incorporated by reference.

[0129] Another maintenance medium is described in U.S. Pat. No.5,096,822 to Rosenkrans, Jr. et al., which is incorporated herein byreference. This embryo medium, named CR1, contains the nutritionalsubstances necessary to support an embryo. CR1 contains hemicalciumL-lactate in amounts ranging from 1.0 mM to 10 mM, preferably 1.0 mM to5.0 mM. Hemicalcium L-lactate is L-lactate with a hemicalcium saltincorporated thereon.

[0130] Also, suitable culture medium for maintaining human embryoniccells in culture as discussed in Thomson et al., Science, 282:1145-1147(1998) and Proc. Natl. Acad. Sci., USA, 92:7844-7848 (1995).

[0131] Afterward, the cultured NT unit or units are preferably washedand then placed in a suitable media, e.g., CR1aa medium, Ham's F-10,Tissue Culture Media-199 (TCM-199). Tyrodes-Albumin-Lactate-Pyruvate(TALP) Dulbecco's Phosphate Buffered Saline (PBS), Eagle's or Whitten's,preferably containing about 10% FCS. Such culturing will preferably beeffected in well plates which contain a suitable confluent feeder layer.Suitable feeder layers include, by way of example, fibroblasts andepithelial cells, e.g., fibroblasts and uterine epithelial cells derivedfrom ungulates, chicken fibroblasts, murine (e.g., mouse or rat)fibroblasts, STO and SI-m220 feeder cell lines, and BRL cells.

[0132] In the preferred embodiment, the feeder cells will comprise mouseembryonic fibroblasts. Means for preparation of a suitable fibroblastfeeder layer are described in the example which follows and is wellwithin the skill of the ordinary artisan.

[0133] In a preferred embodiment the nuclear transfer unit, orblastocysts, morula or inner cell mast or cells derived therefrom, arepermitted to differentiate into desired cell lineages by expression todifferent combinations of growth factors, hormones will be derived fromthe same species as the donor cell or nucleus.

[0134] Also preferably, the growth factors or hormones will berecombinantly produced rather than isolated directly from an animal, toavoid viral contaminants.

[0135] The NT units are preferably cultured on a feeder layer until theNT units reach a size suitable for obtaining cells which may be used toproduce embryonic stem-like cells or cell colonies. Preferably, these NTunits will be cultured until they reach a size of at least about 2 to400 cells, more preferably about 4 to 128 cells, and most preferably atleast about 50 cells. The culturing will be effected under suitableconditions, i.e., about 38.5□C. and 5% CO₂, with the culture mediumchanged in order to optimize growth typically about every 2-5 days,preferably about every 3 days.

[0136] In the case of human cell/enucleated bovine oocyte derived NTunits, sufficient cells to produce an ES cell colony, typically on theorder of about 50 cells, will be obtained about 12 days after initiationof oocyte activation. However, this may vary dependent upon theparticular cell used as the nuclear donor, the species of the particularoocyte, and culturing conditions. One skilled in the art can readilyascertain visually when a desired sufficient number of cells has beenobtained based on the morphology of the cultured NT units.

[0137] In the case of human/human nuclear transfer embryos, or otherembryos produced using non-human primate donor or oocyte, it may beadvantageous to use culture medium known to be useful for maintaininghuman and other primate cells in tissue culture. Examples of a culturemedia suitable for human embryo culture include the medium reported inJones et al, Human Reprod., 13(1):169-177 (1998), the P1-catalog #99242medium, and the P-1 catalog #99292 medium, both available from IrvineScientific, Santa Ana, Calif., and those used by Thomson et al. (1998)and (1995), which references are incorporated by reference in theirentirety.

[0138] Another preferred medium comprises ACM+uridine+glucose+1000 IU ofLIF. As discussed above, the cells used in the present invention willpreferably comprise mammalian somatic cells, most preferably cellsderived from an actively proliferating (non-quiescent) mammalian cellculture. In an especially preferred embodiment, the donor cell will begenetically modified by the addition, deletion or substitution of adesired DNA sequence. For example, the donor cell, e.g., a keratinocyteor fibroblast, e.g., of human, primate or bovine origin, may betransfected or transformed with a DNA construct that provides for theexpression of a desired gene product, e.g., therapeutic polypeptide.Examples thereof include lymphokines, e.g., IGF-II, IGF-II, interferons,colony stimulating factors, connective tissue polypeptides such ascollagens, genetic factors, clotting factors, enzymes, enzymeinhibitors, etc.

[0139] Also, as discussed above, the donor cells may be modified priorto nuclear transfer, e.g., to effect impaired cell lineage development,enhanced embryonic development and/or inhibition of apoptosis. Examplesof desirable modifications are discussed further below.

[0140] One aspect of the invention will involve genetic modification ofthe donor cell, e.g., a human cell, such that it is lineage deficientand therefore when used for nuclear transfer it will be unable to giverise to a viable offspring. This is desirable especially in the contextof human nuclear transfer embryos, wherein for ethical reasons,production of a viable embryo may be an unwanted outcome. This can beeffected by genetically engineering a human cell such that it isincapable of differentiating into specific cell lineages when used fornuclear transfer. In particular, cells may be genetically modified suchthat when used as nuclear transfer donors the resultant “embryos” do notcontain or substantially lack at least one of mesoderm, endoderm orectoderm tissue.

[0141] This can be accomplished by, e.g., knocking out or impairing theexpression of one or more mesoderm, endoderm or ectoderm specific genes.Examples thereof include: Mesoderm: SRF, MESP-1, HNF-4, beta-I integrin,MSD; Endoderm: GATA-6, GATA-4; Ectoderm: RNA helicase A, H beta 58.

[0142] The above list is intended to be exemplary and non-exhaustive ofknown genes which are involved in the development of mesoderm, endodermand ectoderm. The generation of mesoderm deficient, endoderm deficientand ectoderm deficient cells and embryos has been previously reported inthe literature. See, e.g., Arsenian et al, EMBO J., Vol. 17(2):6289-6299(1998); Saga Y, Mech. Dev., Vol. 75(1-2):53-66 (1998); Holdener et al,Development, Vol. 120(5):1355-1346 (1994); Chen et al, Genes Dev. Vol.8(20):2466-2477 (1994); Rohwedel et al, Dev. Biol., 201(2): 167-189(1998) (mesoderm); Morrisey et al, Genes, Dev., Vol.12(22):3579-3590(1998); Soudais et al, Development, Vol. 121(11):3877-3888 (1995)(endoderm); and Lee et al, Proc. Natl. Acad. Sci. USA, Vol.95:(23):13709-13713 (1998); and Radice et al, Development, Vol.111(3):801-811 (1991) (ectoderm).

[0143] In general, a desired somatic cell, e.g., a human keratinocyte,epithelial cell or fibroblast, will be genetically engineered such thatone or more genes specific to particular cell lineages are “knocked out”and/or the expression of such genes significantly impaired. This may beeffected by known methods, e.g., homologous recombination. A preferredgenetic system for effecting “knock-out” of desired genes is disclosedby Capecchi et al, U.S. Pat. Nos. 5,631,153 and 5,464,764, which reportspositive-negative selection (PNS) vectors that enable targetedmodification of DNA sequences in a desired mammalian genome. Suchgenetic modification will result in a cell that is incapable ofdifferentiating into a particular cell lineage when used as a nucleartransfer donor.

[0144] This genetically modified cell will be used to produce alineage-defective nuclear transfer embryo, i.e., that does not developat least one of a functional mesoderm, endoderm or ectoderm. Thereby,the resultant embryos, even if implanted, e.g., into a human uterus,would not give rise to a viable offspring. However, the ES cells thatresult from such nuclear transfer will still be useful in that they willproduce cells of the one or two remaining non-impaired lineage. Forexample, an ectoderm deficient human nuclear transfer embryo will stillgive rise to mesoderm and endoderm derived differentiated cells. Anectoderm deficient cell can be produced by deletion and/or impairment ofone or both of RNA helicase A or H beta 58 genes.

[0145] These lineage deficient donor cells may also be geneticallymodified to express another desired DNA sequence.

[0146] Thus, the genetically modified donor cell will give rise to alineage-deficient blastocyst which, when plated, will differentiate intoat most two of the embryonic germ layers.

[0147] Alternatively, the donor cell can be modified such that it is“mortal”. This can be achieved by expressing anti-sense or ribozymetelomerase genes. This can be effected by known genetic methods thatwill provide for expression of antisense DNA or ribozymes, or by geneknockout. These “mortal” cells, when used for nuclear transfer, will notbe capable of differentiating into viable offspring.

[0148] Another preferred embodiment of the present invention is theproduction of nuclear transfer embryos that grow more efficiently intissue culture. This is advantageous in that it should reduce therequisite time and necessary fusions to produce ES cells and/oroffspring (if the blastocysts are to be implanted into a femalesurrogate). This is desirable also because it has been observed thatblastocysts and ES cells resulting from nuclear transfer may haveimpaired development potential. While these problems may often bealleviated by alteration of tissue culture conditions, an alternativesolution is to enhance embryonic development by enhancing expression ofgenes involved in embryonic development.

[0149] For example, it has been reported that the gene products of thePed type, which are members of the MHC I family, are of significantimportance to embryonic development. More specifically, it has beenreported in the case of mouse preimplantation embryos that the Q7 and Q9genes arereesponsible forthe “fast growth” phenotype. Therefore, it isanticipated that introduction of DNAs that provide for the expression ofthese and related genes, or their human or other mammalian counterpartsinto donor cells, will give rise to nuclear transfer embryos that growmore quickly. This is particularly desirable in the context ofcross-species nuclear transfer embryos which may develop lessefficiently in tissue culture than nuclear transfer embryos produced byfusion of cells or nuclei of the same species.

[0150] In particular, a DNA construct containing the Q7 and/or Q9 genewill be introduced into donor somatic cells prior to nuclear transfer.For example, an expression construct can be constructed containing astrong constitutive mammalian promoter operably linked to the Q7 and/orQ9 genes, an IRES, one or more suitable selectable markers, e.g,.neomycin, ADA, DHFR, and a poly-A sequence, e.g., bGH polyA sequence.Also, it may be advantageous to further enhance Q7 and Q9 geneexpression by the inclusion of insulates. It is anticipated that thesegenes will be expressed early on in blastocyst development as thesegenes are highly conserved in different species, e.g., bovines, goats,rabbits, porcine, dogs, cats, and humans. Also, it is anticipated thatdonor cells can be engineered to affect other genes that enhanceembryonic development. Thus, these genetically modified donor cellsshould produce blastocysts and preimplantation stage embryos moreefficiently.

[0151] Still another aspect of the invention involves the constructionof donor cells that are resistant to apoptosis, i.e., programmed celldeath. It has been reported in the literature that cell death relatedgenes are present in preimplantation stage embryos. (Adams et al,Science, 281(5381):1322-1326 (1998)). Genes reported to induce apoptosisinclude, e.g., Bad, Bok, BH3, Bik, Hrk, BNIP3, Bim_(L), Bad, Bid, andEGL-1. By contrast, genes that reportedly protect cells from programmedcell death include, by way of example, BcL-XL, Bcl-w, Mcl-1, A1, Nr-13,BHRF-1, LMW5-HL, ORF16, Ks-Bel-2, E1B-19K, and CED-9.

[0152] Thus, donor cells can be constructed wherein genes that induceapoptosis are “knocked out” or wherein the expression of genes thatprotect the cells from apoptosis is enhanced or turned on duringembryonic development.

[0153] For example, this can be effected by introducing a DNA constructthat provides for regulated expression of such protective genes, e.g.,Bcl-2 or related genes during embryonic development. Thereby, the genecan be “turned on” by culturing the embryo under specific growthconditions. Alternatively, it can be linked to a constitutive promoter.

[0154] More specifically, a DNA construct containing a Bcl-2 geneoperably linked to a regulatable or constitutive promoter, e.g., PGK,SV40, CMV, ubiquitin, or beta-actin, an IRES, a suitable selectablemarker, and a poly-A sequence can be constructed and introduced into adesired donor mammalian cell, e.g., human keratinocyte or fibroblast.

[0155] These donor cells, when used to produce nuclear transfer embryos,should be resistant to apoptosis and thereby differentiate moreefficiently in tissue culture. Thereby, the speed and/or number ofsuitable preimplantation embryos produced by nuclear transfer can beincreased.

[0156] Another means of accomplishing the same result is to impair theexpression of one or more genes that induce apoptosis. This will beeffected by knock-out or by the use of antisense or ribozymes againstgenes that are expressed in and which induce apoptosis early on inembryonic development. Examples thereof are identified above. Cell deathgenes that may be expressed in the antisense orientation include BAX,Apaf-1, and capsases. Additionally, a transgene may be introduced thatencodes for methylase or demethylase in the sense or antisenseorientation. DNAs that encode methylase and demethylase enzymes are wellknown in the art. Still alternatively, donor cells may be constructedcontaining both modifications, i.e., impairment of apoptosis-inducinggenes and enhanced expression of genes that impede or prevent apoptosis.The construction and selection of genes that affect apoptosis, and celllines that express such genes, is disclosed in U.S. Pat. No. 5,646,008,which patent is incorporated by reference herein. Many DNAs that promoteor inhibit apoptosis have been reported and are the subject of numerouspatents.

[0157] Another means of enhancing cloning efficiency is to select cellsof a particular cell cycle stage as the donor cell. It has been reportedthat this can have significant effects on nuclear transfer efficiency.(Barnes et al, Mol. Reprod. Devel., 36(1):33-41 (1993). Differentmethods for selecting cells of a particular cell cycle stage have beenreported and include serum starvation (Campbell et al, Nature, 380:64-66(1996); Wilmut et al, Nature, 385:810-813 (1997), and chemicalsynchronization (Urbani et al, Exp. Cell Res., 219(1):159-168 (1995).For example, a particular cyclin DNA may be operably linked to aregulatory sequence, together with a detectable marker, e.g., greenfluorescent protein (GFP), followed by the cyclin destruction box, andoptionally insulation sequences to enhance cyclin and marker proteinexpression. Thereby, cells of a desired cell cycle can be easilyvisually detected and selected for use as a nuclear transfer donor. Anexample thereof is the cyclin D1 gene in order to select for cells thatare in G1. However, any cyclin gene should be suitable for use in theclaimed invention. (See, e.g., King et al, Mol. Biol. Cell, Vol.7(9):1343-1357 (1996)).

[0158] However, a less invasive or more efficient method for producingcells of a desired cell cycle stage are needed. It is anticipated thatthis can be effected by genetically modifying donor cells such that theyexpress specific cyclins under detectable conditions. Thereby, cells ofa specific cell cycle can be readily discerned from other cell cycles.

[0159] Cyclins are proteins that are expressed only during specificstages of the cell cycle. They include cyclin D1, D2 and D3 in G1 phase,cyclin B1 and B2 in G2/M phase and cyclin E, A and H in S phase. Theseproteins are easily translated and destroyed in the cytogolcytosol. This“transient” expression of such proteins is attributable in part to thepresence of a “destruction box”, which is a short amino acid sequencethat is part of the protein that functions as a tag to direct the promptdestruction of these proteins via the ubiquitin pathway. (Adams et al,Science, 281 (5321):1322-1326 (1998)).

[0160] In the present invention, donor cells will be constructed thatexpress one or more of such cyclin genes under easily detectableconditions, preferably visualizable, e.g., by the use of a fluorescentlabel. For example, a particular cyclin DNA may be operably linked to aregulatory sequence, together with a detectable marker, e.g., greenfluorescent protein (GFP), followed by the cyclin destruction box, andoptionally insulation sequences to enhance cyclin and/or marker proteinexpression. Thereby, cells of a desired cell cycle can be easilyvisually detected and selected for use as a nuclear transfer donor. Anexample thereof is the cyclin D1 gene which can be used to select forcells that are in G1. However, any cyclin gene should be suitable foruse in the claimed invention. (See, e.g., King et al, Mol. Biol. Cell,Vol. 7(9):1343-1357 (1996)).

[0161] As discussed, the present invention provides different methodsfor enhancing nuclear transfer efficiency, preferably a cross-speciesnuclear transfer process. While the present inventors have demonstratedthat nuclei or cells of one species when inserted or fused with anenucleated oocyte of a different species can give rise to nucleartransfer embryos that produce blastocysts, which embryos can give riseto ES cell lines, the efficiency of such process is lower than for somespecies donor cell/nucleus/recipient transfer Therefore, many fusionstypically need to be effected to produce a blastocyst the cells of whichmay be cultured to produce ES cells and ES cell lines. Yet another meansfor enhancing the development of nuclear transfer embryos in vitro is byoptimizing culture conditions. One means of achieving this result willbe to culture NT embryos under conditions impede apoptosis. With respectto this embodiment of the invention, it has been found that proteasessuch as capsases can cause oocyte death by apoptosis similar to othercell types. (See, Jurisicosva et al, Mol. Reprod. Devel., 51(3):243-253(1998).)

[0162] It is anticipated that blastocyst development will be enhanced byincluding in culture media used for nuclear transfer and to maintainblastocysts or culture pre-implantation stage embryos one or morecapsase inhibitors. Such inhibitors include by way of example capsase-4inhibitor I, capsase-3 inhibitor I, capsase-6 inhibitor II, capsase-9inhibitor II, and capsase-1 inhibitor I. The amount thereof will be anamount effective to inhibit apoptosis, e.g., 0.00001 to 5.0% by weightof medium; more preferably 0.01 % to 1.0% by weight of medium. Thus, theforegoing methods may be used to increase the efficiency of nucleartransfer by enhancing subsequent blastocyst and embryo development intissue culture.

[0163] After NT units of the desired size are obtained, the cells aremechanically removed from the zone and are then used to produceembryonic or stem-like cells and cell lines. This is preferably effectedby taking the clump of cells which comprise the NT unit, which typicallywill contain at least about 50 cells, washing such cells, and platingthe cells onto a feeder layer, e.g., irradiated fibroblast cells.Typically, the cells used to obtain the stem-like cells or cell colonieswill be obtained from the inner most portion of the cultured NT unitwhich is preferably at least 50 cells in size. However, NT units ofsmaller or greater cell numbers as well as cells from other portions ofthe NT unit may also be used to obtain ES-like cells and cell colonies.

[0164] It is further envisioned that a longer exposure of donor cell DNAto the oocyte's cytosol may facilitate the dedifferentiation process.This can be accomplished by re-cloning, i.e., by taking blastomeres froma reconstructed embryo and fusing them with a new enucleated oocyte.Alternatively, the donor cell may be fused with an enucleated oocyte andfourto six hours later, without activation, chromosomes removed andfused or injected into another, e.g., younger oocyte. Activation wouldoccur thereafter.

[0165] The cells are maintained in the feeder layer in a suitable growthmedium, e.g., alpha MEM supplemented with 10% FCS and 0.1 mMbeta-mercaptoethanol (Sigma) and L-glutamine. The growth medium ischanged as often as necessary to optimize growth, e.g., about every 2-3days.

[0166] This culturing process results in the formation of embryonic orstem-like cells or cell lines. In the case of human cell/bovine oocytederived NT embryos, colonies are observed by about the second day ofculturing in the alpha MEM medium. However, this time may vary dependentupon the particular nuclear donor cell, specific oocyte and culturingconditions. One skilled in the art can vary the culturing conditions asdesired to optimize growth of the particular embryonic or stem-likecells. Other suitable media are disclosed herein.

[0167] The embryonic or stem-like cells and cell colonies obtained willtypically exhibit an appearance similar to embryonic or stem-like cellsof the species used as the nuclear cell donor rather than the species ofthe donor oocyte. For example, in the case of embryonic or stem-likecells obtained by the transfer of a human nuclear donor cell into anenucleated bovine oocyte, the cells exhibit a morphology more similar tomouse embryonic stem cells than bovine ES-like cells.

[0168] More specifically, the individual cells of the human ES-line cellcolony are not well defined, and the perimeter of the colony isrefractive and smooth in appearance. Further, the cell colony has alonger cell doubling time, about twice that of mouse ES cells. Also,unlike bovine and porcine derived ES cells, the colony does not possessan epithelial-like appearance.

[0169] As discussed above, it has been reported by Thomson, in U.S. Pat.No. 5,843,780, that primate stem cells are SSEA-1 (−), SSEA-4 (+),TRA-1-60 (+), TRA-1-81 (+) and alkaline phosphatase (+). It isanticipated that human and primate ES cells produced according to thepresent methods will exhibit similar or identical marker expression.

[0170] Alternatively, that such cells are actual human or primateembryonic stem cells will be confirmed based on their capability ofgiving rise to all of mesoderm, ectoderm and endoderm tissues. This willbe demonstrated by culturing ES cells produced according to theinvention under appropriate conditions, e.g., as disclosed by Thomsen,U.S. Pat. No. 5,843,780, incorporated by reference in its entiretyherein. Alternatively, the fact that the cells produced according to theinvention are pluripotent will be confirmed by injecting such cells intoan animal, e.g., a SCID mouse, or large agricultural animal, andthereafter obtaining tissues that result from said implanted cells.These implanted ES cells should give rise to all different types ofdifferentiated tissues, i.e., mesoderm, ectoderm, and endodermaltissues.

[0171] The resultant embryonic or stem-like cells and cell lines,preferably human embryonic or stem-like cells and cell lines, havenumerous therapeutic and diagnostic applications. Most especially, suchembryonic or stem-like cells may be used for cell transplantationtherapies. Human embryonic or stem-like cells have application in thetreatment of numerous disease conditions.

[0172] Still another object of the present invention is to improve theefficacy of nuclear transfer, e.g., cross-species nuclear transfer byintroducing mitochondrial DNA of the same species as the donor cell ornucleus into the recipient oocyte before or after nuclear transfer,before or after activation, and before or after fusion and cleavage.Preferably, if the donor cell is human, human mitochondrial DNA will bederived from cells of the particular donor, e.g., liver cells andtissue.

[0173] Methods for isolating mitochondria are well known in the art.Mitochondria can be isolated from cells in tissue culture, or fromtissue. The particular cells or tissue will depend upon the particularspecies of the donor cell. Examples of cells or tissues that may be usedas sources of mitochondria include fibroblasts, epithelium, liver, lung,keratinocyte, stomach, heart, bladder, pancreas, esophageal,lymphocytes, monocytes, mononuclear cells, cumulus cells, uterine cells,placental cells, intestinal cells, hematopoietic cells, and tissuescontaining such cells.

[0174] For example, mitochondria can be isolated from tissue culturecells and rat liver. It is anticipated that the same or similarprocedures may be used to isolate mitochondria from other cells andtissues. As noted above, preferred source of mitochondria compriseshuman liver tissue because such cells contain a large number ofmitochondria. Those skilled in the art will be able to modify theprocedure as necessary, dependent upon the particular cell line ortissue. The isolated DNA can also be further purified, if desired, knownmethods, e.g., density gradient centrifugation.

[0175] In this regard, it is known that mouse embryonic stem (ES) cellsare capable of differentiating into almost any cell type, e.g.,hematopoietic stem cells. Therefore, human embryonic or stem-like cellsproduced according to the invention should possess similardifferentiation capacity. The embryonic or stem-like cells according tothe invention will be induced to differentiate to obtain the desiredcell types according to known methods. For example, the subject humanembryonic or stem-like cells may be induced to differentiate intohematopoietic stem cells, muscle cells, cardiac muscle cells, livercells, cartilage cells, epithelial cells, urinary tract cells, etc., byculturing such cells in differentiation medium and under conditionswhich provide for cell differentiation. Medium and methods which resultin the differentiation of embryonic stem cells are known in the art asare suitable culturing conditions.

[0176] For example, Palacios et al, Proc. Natl. Acad. Sci., USA,92:7530-7537 (1995) teaches the production of hematopoietic stem cellsfrom an embryonic cell line by subjecting stem cells to an inductionprocedure comprising initially culturing aggregates of such cells in asuspension culture medium lacking retinoic acid followed by culturing inthe same medium containing retinoic acid, followed by transferral ofcell aggregates to a substrate which provides for cell attachment.

[0177] Moreover, Pedersen, J. Reprod. Fertil. Dev., 6:543-552 (1994) isa review article which references numerous articles disclosing methodsfor in vitro differentiation of embryonic stem cells to produce variousdifferentiated cell types including hematopoietic cells, muscle, cardiacmuscle, nerve cells, among others.

[0178] Further, Bain et al, Dev. Biol., 168:342-357 (1995) teaches invitro differentiation of embryonic stem cells to produce neural cellswhich possess neuronal properties. These references are exemplary ofreported methods for obtaining differentiated cells from embryonic orstem-like cells. These references and in particular the disclosurestherein relating to methods for differentiating embryonic stem cells areincorporated by reference in their entirety herein.

[0179] Thus, using known methods and culture medium, one skilled in theart may culture the subject embryonic or stem-like cells embryos,cultured inner cell, cell masses, morula, or blastocysts provided bycross-species nuclear transfer to obtain desired differentiated celltypes, e.g., neural cells, muscle cells, hematopoietic cells, etc. Inaddition, the use of inducible Bcl-2 or Bcl-xl might be useful forenhancing in vitro development of specific cell lineages. In vivo, Bcl-2prevents many, but not all, forms of apoptotic cell death that occurduring lymphoid and neural development. A thorough discussion of howBcl-2 expression might be used to inhibit apoptosis of relevant celllineages following transfection of donor cells is disclosed in U.S. Pat.No. 5,646,008, which is herein incorporated by reference.

[0180] The subject embryonic or stem-like cells may be used to obtainany desired differentiated cell type. Therapeutic usages of suchdifferentiated human cells are unparalleled. For example, humanhematopoietic stem cells may be used in medical treatments requiringbone marrow transplantation. Such procedures are used to treat manydiseases, e.g., late stage cancers such as ovarian cancer and leukemia,as well as diseases that compromise the immune system, such as AIDS.Hematopoietic stem cells can be obtained, e.g., by fusing adult somaticcells of a cancer or AIDS patient, e.g., epithelial cells or lymphocyteswith an enucleated oocyte, e.g., bovine oocyte, obtaining embryonic orstem-like cells as described above, and culturing such cells underconditions which favor differentiation, until hematopoietic stem cellsare obtained. Such hematopoietic cells may be used in the treatment ofdiseases including cancer and AIDS.

[0181] Alternatively, adult somatic cells from a patient with aneurological disorder may be fused with an enucleated animal oocyte,e.g., a primate or bovine oocyte, human embryonic or stem-like cellsobtained therefrom, and such cells cultured under differentiationconditions to produce neural cell lines. Specific diseases treatable bytransplantation of such human neural cells include, by way of example,Parkinson's disease, Alzheimers disease, ALS and cerebral palsy, amongothers. In the specific case of Parkinson's disease, it has beendemonstrated that transplanted fetal brain neural cells make the properconnections with surrounding cells and produce dopamine. This can resultin long-term reversal of Parkinson's disease symptoms.

[0182] To allow for specific selection of differentiated cells, donorcells may be transfected with selectable markers expressed via induciblepromoters, thereby permitting selection or enrichment of particular celllineages when differentiation is induced. For example, CD34-neo may beused for selection of hematopoietic cells, Pw1-neo for muscle cells,Mash-1-neo for sympathetic neurons, Mal-neo for human CNS neurons of thegrey matter of the cerebral cortex, etc.

[0183] The great advantage of the subject invention is that it providesan essentially limitless supply of isogenic or synegenic human cellssuitable for transplantation. Therefore, it will obviate the significantproblem associated with current transplantation methods, i.e., rejectionof the transplanted tissue which may occur because of host-vs-graft orgraft-vs-host rejection. Conventionally, rejection is prevented orreduced by the administration of anti-rejection drugs such ascyclosporin. However, such drugs have significant adverse side-effects,e.g., immunosuppression, carcinogenic properties, as well as being veryexpensive. The present invention should eliminate, or at least greatlyreduce, the need for anti-rejection drugs, such as cyclosporine, imulan,FK-506, glucocorticoids, and rapamycin, and derivatives thereof.

[0184] Other diseases and conditions treatable by isogenic cell therapyinclude, byway of example, spinal cord injuries, multiple sclerosis,muscular dystrophy, diabetes, liver diseases, i.e.,hypercholesterolemia, heart diseases, cartilage replacement, bums, footulcers, gastrointestinal diseases, vascular diseases, kidney disease,urinary tract disease, and aging related diseases and conditions.

[0185] Also, human embryonic or stem-like cells produced according tothe invention may be used to produce genetically engineered ortransgenic human differentiated cells. Essentially, this will beeffected by introducing a desired gene or genes, which may beheterologous, or removing all or part of an endogenous gene or genes ofhuman embryonic or stem-like cells produced according to the invention,and allowing such cells to differentiate into the desired cell type. Apreferred method for achieving such modification is by homologousrecombination because such technique can be used to insert, delete ormodify a gene or genes at a specific site or sites in the stem-like cellgenome.

[0186] This methodology can be used to replace defective genes, e.g.,defective immune system genes, cystic fibrosis genes, or to introducegenes which result in the expression of therapeutically beneficialproteins such as growth factors, lymphokines, cytokines, enzymes, etc.For example, the gene encoding brain derived growth factor may beintroduced into human embryonic or stem-like cells, the cellsdifferentiated into neural cells and the cells transplanted into aParkinson's patient to retard the loss of neural cells during suchdisease.

[0187] Previously, cell types transfected with BDNF varied from primarycells to immortalized cell lines, either neural or non-neural (myoblastand fibroblast) derived cells. For example, astrocytes have beentransfected with BDNF gene using retroviral vectors, and the cellsgrafted into a rat model of Parkinson's disease (Yoshimoto et al., BrainResearch, 691:25-36, (1995)).

[0188] This ex vivo therapy reduced Parkinson's-like symptoms in therats up to 45% 32 days after transfer. Also, the tyrosine hydroxylasegene has been placed into astrocytes with similar results (Lundberg etal., Develop. Neurol., 139:39-53 (1996) and references cited therein).

[0189] However, such ex vivo systems have problems. In particular,retroviral vectors currently used are down-regulated in vivo and thetransgene is only transiently expressed (review by Mulligan, Science,260:926-932 (1993)). Also, such studies used primary cells, astrocytes,which have finite life span and replicate slowly. Such propertiesadversely affect the rate of transfection and impede selection of stablytransfected cells. Moreover, it is almost impossible to propagate alarge population of gene targeted primary cells to be used in homologousrecombination techniques.

[0190] By contrast, the difficulties associated with retroviral systemsshould be eliminated by the use of human embryonic or stem-like cells.It has been demonstrated previously by the subject assignee that cattleand pig embryonic cell lines can be transfected and selected for stableintegration of heterologous DNA. Such methods are described in commonlyassigned U.S. Ser. No. 08/626,054, filed Apr. 1, 1996, now U.S. Pat. No.5,905,042, incorporated by reference in its entirety. Therefore, usingsuch methods or other known methods, desired genes may be introducedinto the subject human embryonic or stem-like cells, and the cellsdifferentiated into desired cell types, e.g., hematopoietic cells,neural cells, pancreatic cells, cartilage cells, etc.

[0191] Genes which may be introduced into the subject embryonic orstem-like cells include, by way of example, epidermal growth factor,basic fibroblast growth factor, glial derived neurotrophic growthfactor, insulin-like growth factor (I and II), neurotrophin-3,neurotrophin4/5, ciliary neurotrophic factor, AFT-1, cytokine genes(interleukins, interferons, colony stimulating factors, tumor necrosisfactors (alpha and beta), etc.), genes encoding therapeutic enzymes,collagen, human serum albumin, etc.

[0192] In addition, it is also possible to use one of the negativeselection systems now known in the art for eliminating therapeutic cellsfrom a patient if necessary. For example, donor cells transfected withthe thymidine kinase (TK) gene will lead to the production of embryoniccells containing the TK gene. Differentiation of these cells will leadto the isolation of therapeutic cells of interest which also express theTK gene. Such cells may be selectively eliminated at any time from apatient upon gancyclovir administration. Such a negative selectionsystem is described in U.S. Pat. No. 5,698,446, and is hereinincorporated by reference.

[0193] The subject embryonic or stem-like cells, preferably human cells,also may be used as an in vitro model of differentiation, in particularfor the study of genes which are involved in the regulation of earlydevelopment.

[0194] Also, differentiated cell tissues and organs using the subjectembryonic or stem-like cells may be used in drug studies.

[0195] Further, the subject cells may be used to express recombinantDNAs.

[0196] Still further, the subject embryonic or stem-like cells may beused as nuclear donors for the production of other embryonic orstem-like cells and cell colonies.

[0197] Also, cultured inner cell mass, or stem cells, produced accordingto the invention may be introduced into animals, e.g., SCID mice, cows,pigs, e.g., under the renal capsule or intramuscularly and used toproduce a teratoma therein. This teratoma can be used to derivedifferent tissue types. Also, the inner cell mass produced by X-speciesnuclear transfer may be introduced together with a biodegradable,biocompatible polymer matrix that provides for the formation of3-dimensional tissues. After tissue formation, the polymer degrades,ideally just leaving the donor tissue, e.g., cardiac, pancreatic,neural, lung, liver. In some instances, it may be advantageous toinclude growth factors and proteins that promote angiogenesis.Alternatively, the formation of tissues can be effected totally invitro, with appropriate culture media and conditions, growth factors,and biodegradable polymer matrices. The invention further encompassesscreening different combinations of hormones and/or growth factors toidentify those that induce the differentiation of cultured inner cellmasses, morula, blastocysts or cells derived therefrom into desired celllineages. Alternatively, differentiation may take place spontaneouslyupon removal of a cross-species embryo provided according to theinvention from a future layer.

[0198] In order to more clearly describe the subject invention, thefollowing examples are provided.

EXAMPLE 1 Materials and Methods

[0199] Donor Cells for Nuclear Transfer

[0200] Epithelial cells were lightly scraped from the inside of themouth of a consenting adult with a standard glass slide. The cells werewashed off the slide into a petri dish containing phosphate bufferedsaline without Ca or Mg. The cells were pipetted through a small-borepipette to break up cell clumps into a single cell suspension. The cellswere then transferred into a microdrop of TL-HEPES medium containing 10%fetal calf serum (FCS) under oil for nuclear transfer into enucleatedcattle oocytes.

[0201] Nuclear Transfer Procedures

[0202] Basic nuclear transfer procedures have been described previously.Briefly, after slaughterhouse oocytes were matured in vitro the oocyteswere stripped of cumulus cells and enucleated with a beveledmicropipette at approximately 18 hours post maturation (hpm).Enucleation was confirmed in TL-HEPES medium plus bisbenzimide (Hoechst33342, 3 μg/ml; Sigma). Individual donor cells were then placed into theperivitelline space of the recipient oocyte. The bovine oocyte cytoplasmand the donor nucleus (NT unit) are fused together using electrofusiontechniques. One fusion pulse consisting of 90 V for 15 μsec was appliedto the NT unit. This occurred at 24 hours post-initiation of maturation(hpm) of the oocytes. The NT units were placed in CR1aa medium until 28hpm.

[0203] The procedure used to artificially activate oocytes has beendescribed elsewhere. NT unit activation was at 28 hpm. A briefdescription of the activation procedure is as follows: NT units wereexposed for four min to ionomycin (5 μM; CalBiochem, La Jolla, Calif.)in TL-HEPES supplemented with 1 mg/ml BSA and then washed for five minin TL-HEPES supplemented with 30 mg/ml BSA. The NT units were thentransferred into a microdrop of CR1aa culture medium containing 0.2 mMDMAP (Sigma) and cultured at 38.5° C. 5% CO₂ for four to five hours. TheNT units were washed and then placed in a CR1aa medium plus 10% FCS and6 mg/ml BSA in four well plates containing a confluent feeder layer ofmouse embryonic fibroblasts (described below). The NT units werecultured for three more days at 38.5° C. and 5% CO₂. The culture mediumwas changed every three days until day 12 after the time of activation.At this time NT units reaching the desired cell number, i.e., about 50cell number, were mechanically removed from the zona and used to produceembryonic cell lines. A photograph of an NT unit obtained as describedabove is contained in FIG. 1.

[0204] Fibroblast Feeder Layer

[0205] Primary cultures of embryonic fibroblasts were obtained from14-16 day old murine fetuses. After the head, liver, heart andalimentary tract were aseptically removed, the embryos were minced andincubated for 30 minutes at 37° C. in pre-warmed trypsin EDTA solution(0.05% trypsin/0.02% EDTA; GIBCO, Grand Island, N.Y.). Fibroblast cellswere plated in tissue culture flasks and cultured in alpha-MEM medium(BioWhittaker, Walkersville, Md.) supplemented with 10% fetal calf serum(FCS) (Hyclone, Logen, Utah), penicillin (100 IU/ml) and streptomycin(50 μl/ml). Three to four days after passage, embryonic fibroblasts, in35×10 Nunc culture dishes (Baxter Scientific, McGaw Park, Ill.), wereirradiated. The irradiated fibroblasts were grown and maintained in ahumidified atmosphere with 5% CO₂ in air at 37° C. The culture plateswhich had a uniform monolayer of cells were then used to cultureembryonic cell lines.

[0206] Production of Embryonic Cell Line.

[0207] NT unit cells obtained as described above were washed and plateddirectly onto irradiated feeder fibroblast cells. These cells includedthose of the inner portion of the NT unit. The cells were maintained ina growth medium consisting of alpha MEM supplemented with 10% FCS and0.1 mM beta-mercaptoethanol (Sigma). Growth medium was exchanged everytwo to three days. The initial colony was observed by the second day ofculture. The colony was propagated and exhibits a similar morphology topreviously disclosed mouse embryonic stem (ES) cells. Individual cellswithin the colony are not well defined and the perimeter of the colonyis refractile and smooth in appearance. The cell colony appears to havea slower cell doubling time than mouse ES cells. Also, unlike bovine andporcine derived ES cells, the colony does not have an epithelialappearance thus far. FIGS. 2 through 5 are photographs of ES-like cellcolonies obtained as described, supra.

[0208] Production of Differentiated Human Cells

[0209] The human embryonic cells obtained are transferred to adifferentiation medium and cultured until differentiated human celltypes are obtained.

Results

[0210] Table 1. Human cells as donor nuclei in NT unit production anddevelopment. TABLE 1 No. NT No. NT units 2 No. NT units to 4-16 No. NTunits to 16 - Cell type units made cell stage (%) cell stage (%) 400cell stage (%) lymphocytes 18 12 (67%) 3 (17%) 0 oral cavity epithelium34 18 (53%) 3 (9%) 1 (3%) adult fibroblasts 46  4 (9%) 12 (4 cell; 26%)—  8 (8-16 cells; 17.4%)

[0211] The one NT unit that developed a structure having greater than 16cells was plated down onto a fibroblast feeder layer. This structure wasattached to the feeder layer and started to propagate forming a colonywith a ES cell-like morphology (See, e.g., FIG. 2). Moreover, althoughthe 4 to 16 cell stage structures were not used to try and produce an EScell colony, it has been previously shown that this stage is capable ofproducing ES or ES-like cell lines (mouse, Eistetter et al., DevelGrowth and Differ., 31:275-282 (1989); Bovine, Stice et al., 1996)).Therefore, it is expected that 4-16 cell stage NT units should also giverise to embryonic or stem-like cells and cell colonies.

[0212] Also, similar results were obtained upon fusion of an adult humankeratinocyte cell line with an enucleated bovine oocyte, which wascultured in media comprising ACM, uridine, glucose, and 1000 IU of LIF.Out of 50 reconstructed embryos, 22 cleaved and one developed into ablastocyst at about day 12. This blastocyst was plated and theproduction of an ES cell line is ongoing.

EXAMPLE 2

[0213] A. Mitochondria Isolation Protocol from a Cell

[0214] This Example relates to isolation of mitochondria and use thereofto enhance the efficiency of cross-species nuclear transfer. The numberof mitochondria per cell varies from cell line to cell line. Forexample, mouse L cells contain ˜100 mitochondria per cell, whereas thereare at least twice that number in HeLa cells. The cells are swollen in ahypotonic buffer and ruptured with a few strokes in a Dounce homogenizerusing a tight-fitting pestle, and the mitochondria are isolated bydifferential centrifugation.

[0215] The solutions, tubes, and homogenizer should be pre-chilled onice. All centrifugation steps are at 40□C. This protocol is based onstarting with a washed cell pellet of 1-2 ml. The cell pellet isresuspended in 11 ml of ice-cold RSB and transferred to a 16 ml Douncehomogenizer.

[0216] RSB Buffer

[0217] RSB (A hypotonic buffer for swelling the tissue culture cells)

[0218] 10 mM NaCl

[0219] 1.5 mM MgCl₂

[0220] 10 mM Tris-HCl, pH 7.5

[0221] MgCl₂

[0222] The cells are allowed to swell for five to ten minutes. Theprogress of the swelling is maintained using a phase contrastmicroscope. The swollen cells are replaced, preferably by severalstrokes with a pestle. Immediately after, 8 ml of 2.5×MS buffer areadded to give a final concentration of 1×MS. The top of the homogenizeris then covered with Parafilm and mixed by inverting a couple of times.

[0223] 2.5×MS Buffer

[0224] 525 mM mannitol

[0225] 175 Mm sucrose

[0226] 12.5 mM ris-HCl, pH 7.5

[0227] 215 mM EDTA pH 7.5

[0228] 1×MS Buffer

[0229] 210 mM mannitol

[0230] 70 mM Sucrose

[0231] 5 mM Tris-HCl, pH 7.5

[0232] 1 mM EDTA, pH 7.5

[0233] MS Buffer is an iso-osmotic buffer to maintain the tonicity ofthe organelles and prevent agglutination.

[0234] Thereafter, the homogenate is transferred to a centrifuge tubefor differential centrifugation. The homogenizer is rinsed with a smallamount of MS buffer and added to the homogenate. The volume is broughtto 30 ml with MS buffer. The homogenate is then centrifuged at 1300 gfor five minutes to remove nuclei, unbroken cells, and large membranefragments. The supernatant is then poured into a clean centrifuge tube.The nuclear spin-down is repeated twice. The supernatant is thentransferred to a clean centrifuge tube and a pellet containing themitochondria is centrifuged at 17,000 g for 15 minutes. The supernatantis discarded and the inside of the tube wiped with a Kimwipe. Themitochondria is washed by re-suspending the pellet in 1×MS and repeatingthe 17,000 g sedimentation. The supernatant is discarded and the pelletis resuspended in a buffer. Mitochondria can be stored at −80□C. forprolonged periods, e.g., up to a year, but preferably will be usedshortly thereafter for NT.

[0235] This basic protocol can be modified. In particular, it may bedesirable to further isolate mitochondrial DNA and us same for NT. Insuch case, contamination with nuclei, not small organelles, potentiallyis a problem and the following modifications may be made. For example,the cells may be harvested in stationary growth phase when the fewestcells are actively dividing, and CaCl₂ substituted for MgCl₂ in the RSBto stabilize the nuclear membrane. The washing of the mitochondrialpellet is omitted as is the density gradient purification. Instead, themitochondrial pellet is simply resuspended and lysed, and themitochondrial DNA purified from any remaining nuclear DNA. As noted,suitable methods for purifying mitachondria and mitochondrial DNA arewell known in the art.

[0236] Homogenization works best if the cells are resuspended in atleast 5-10× the volume of the cell pellet and if the cell suspensionfills the homogenizer at least half full. Press the homogenizer pestlestraight down the tube, maintaining a firm, steady pressure. The Douncehomogenizer disrupts swollen tissue culture cells by pressure change. Asthe pestle is pressed down, pressure around the cell increases; when thecell slips past the end of the pestle, the sudden decrease in pressurecauses the cell to rupture. If the pestle is very tight fitting, theremay be some mechanical breakage as well.

[0237] B. Isolation of Mitochondria from Tissue

[0238] A mitochondrial isolation protocol is selected based on theparticular tissue. For example, the homogenization buffer should beoptimized for the tissue, and the optimal way to homogenize the tissueutilized. Suitable methods are well known in the art.

[0239] Rat liver is the most frequently used tissue for mitochondrialpreparations because it is readily available, is easy to homogenize, andthe cells contain a large number of mitochondria (1000-2000 per cell).For example, a motor-driven, Teflon and glass Potter-Elvehjemhomogenizer can be used homogenize rat liver. Alternatively, if thetissue is soft enough, a Dounce homogenizer with a loose pestle can beused. The yield and purity of the mitochondrial preparation isinfluenced by the method of preparation, speed of preparation, and theage and physiological condition of the animal. As noted, methods ofpurifying mitochondria are well known.

[0240] Preferably, the buffer, tubes, and homogenizer will bepre-chilled. Pre-chilling a glass and Teflon type homogenizer createsthe proper gap between the tube and pestle. The centrifugation steps arepreferably effected at 40□C.

[0241] Essentially, the process will comprise removal of the liver,taking care not to rupture the gall bladder. This is placed in a beakeron ice and any connective tissue is removed. The tissue is recognizedand returned to the beaker, e.g., using very sharp scissors, a scalpel,or razor blade, mince it into 1-2 slices. The pieces are then rinsed,preferably twice, with homogenization buffer (1×MS) to remove most ofthe blood, and the tissue transferred to the homogenizer tube. Enoughhomogenization buffer if added to prepare a 1:10 (w/v) homogenate.

[0242] Use of Isolated Mitochondria or Mitochondrial DNA to Enhance NTEfficacy

[0243] It is theorized by the inventors that the efficacy ofcross-species nuclear transfer may be enhanced by introduction ofmitochondria or mitochondrial DNA at the same species as donor cell ornucleus. Thereupon, the nucleus DNA of resultant NT units will bespecies compatible.

[0244] Mitochondria isolated by the above or other known procedures areincorporated, typically by injection, into any of the following (in thecase of human donor cell/bovine oocyte nuclear transfer):

[0245] (i) non-activated, non-enucleated bovine oocytes;

[0246] (ii) non-activated, enucleated bovine oocytes;

[0247] (iii) activated, enucleated bovine oocytes;

[0248] (iv) non-activated, fused (with human donor cell or nucleus)bovine oocytes;

[0249] (v) activated, fused and cleaved reconstructed (cow oocyte/humancell) embryo; or

[0250] (vi) activated, fused one cell reconstructed (cow oocyte/humancell) embryo.

[0251] The same procedures will enhance other cross-species NTs.Essentially, mitochondria will again be introduced into any of (i)-(vi)of the same species as the donor cell or nucleus, and the oocyte will beof a different species origin. Generally about 1 to 200 picoliters ofmitochondrial suspension are injected into any of the above. Theintroduction of such mitochondria will result in NT units wherein themitochondrial and donor DNA are compatible.

EXAMPLE 3

[0252] Another method for improving the efficacy of the cross-speciesnuclear transfer comprises the fusion of one or more enucleated somaticcells, typically human (of the same species as donor cell or nucleus),with any of the following:

[0253] (i) non-activated, non-enucleated (e.g., bovine) oocyte;

[0254] (ii) non-activated, enucleated (e.g., bovine) oocyte;

[0255] (iii) activated, enucleated (e.g., bovine) oocyte;

[0256] (iv) non-activated, fused (with human cell) oocyte (typicallybovine);

[0257] (v) activated, fused and cleaved reconstructed (e.g., cowoocyte/human cell) embryo;

[0258] (vi) activated, fused one cell reconstructed (cow oocyte/humancell) embryo; or

[0259] (vii) non-activated, fused (e.g., with human cell) oocyte(typically bovine oocyte).

[0260] Fusion is preferably effected by electrical pulse or by use ofSendai virus. Methods for producing enucleated cells (e.g., human cells)are known in the art. A preferred protocol is set forth below.

[0261] Enucleation Procedures:

[0262] Methods for the large-scale enucleation of cells withcytochalasin B are well known in the art. Enucleation is preferablyeffected using the monolayer technique. This method uses small numbersof cells attached to the growth surface of a culture disc and is idealif limited numbers of donor cells are available. Another suitableprocedure, the gradient technique, requires centrifugation of cellsthrough Ficoll gradients and is best suited for enucleation of largenumber (>10⁷) of cells.

[0263] Monolayer Technique. The monolayer technique is ideal forvirtually any cell which grows attached to the growth surface.

[0264] Polycarbonate or polypropylene 250-ml wide-mouth centrifugebottles with screw-top caps are sterilized by autoclaving. The capspreferably are autoclaved separately from the bottle to prevent damageto the centrifuge bottle. The bottle are prepared for the enucleationprocedure by the sterile addition of 30 ml DMEM, 2 ml bovine serum, and0.32 ml cytochalasin B (1 mg/ml) to each. The caps are placed on thebottles, and the bottles are maintained at 370° prior to use.

[0265] The cells to be enucleated (from a few hundred to ˜10⁵ cells) areseeded on a culture dish (35×15 mm; Nunc Inc., Naperville, Ill.).Typically, the cells are grown for at least twenty-four hours on thedishes to promote maximal attachment to the growth surface. Preferably,the cells are prevented from becoming confluent. The culture dish isprepared for centrifugation by wiping the outside of the bottom half ofthe dish (containing the cells) with 70% (v/v) ethanol for the purposeof sterilization. Alternatively, the dish can be kept sterile duringcell culturing by maintaining it within a larger, sterile culture dish.The medium is removed from the dish and the dish (without top) is placedupside down within the centrifuge bottle.

[0266] The rotor (GSA, DuPont, Wilmington, Del.) and centrifuge arepreferably pre-warmed to 37° by centrifugation for 30-45 minutes at 8000rpm. The HS-4 swinging-bucket rotor (DuPont) can alternatively be used.The optimal time and speed of centrifugation varies for each cell type.For myoblasts and fibroblasts, the centrifuge bottle with the culturedish is placed in the pre-warmed rotor and centrifuged for approximately20 minutes (interval between the time when the rotor reaches the desiredspeed and the time when the centrifuge is turned off). Preferably,speeds of 6500 to 7200 rpm are used.

[0267] After centrifugation, the centrifuge bottle is removed from therotor, and the culture plate is removed from the bottles with forceps. Asmall amount of medium is maintained in the plate to keep the cellsmoist in order to maintain cell viability. The outside of the dish,including the top edge, is wiped with a sterile wiper, then moistenedwith 95% (v/v) ethanol, to remove any medium and to dry it. A steriletop is placed onto the dish. If the enucleated cells are not going to beused immediately, complete culture medium (medium supplemented with theappropriate concentration of serum) should be added to the dish, and thecells placed in a CO₂ incubator. The resultant enucleated cells(karyoplast) are fused with any of (i)-(viii) above.

[0268] While the present invention has been described and illustratedherein by reference to various specific materials, procedures, andexamples, it is understood that the invention is not restricted to theparticular material, combinations of materials, and procedures selectedfor that purpose. Numerous variations of such details can be implied andwill be appreciated by those skilled in the art.

EXAMPLE 4

[0269] Protocol for the Derivation of Human Stem Cells using CrossSpecies Nuclear Transfer with Rabbit Eggs and Human Somatic Cells

[0270] Superovulation:

[0271] 1—Super ovulate New Zealand does age 8-12 months usingconsecutive injections of FSH, the first two 0.3 mgr and the last four,0.4 mgr twelve hours apart.

[0272] 2—12 hrs after the last FSH injection, a single i.v. injection of100 IU of hCG is given.

[0273] Oocyte Collection:

[0274] 1—12 to 24 hrs after hCG injection, flush oviducts using warm(37° C.) DPBS+1% bovine serum albumin (BSA) or HTF with 1% human serumalbumin (HTF-HSA). 2—Search eggs in a dissecting microscope and placedin transport media—DPBS+1% bovine serum albumin (BSA) or HTF with 1% BSA(HTF-BSA). 3—Transport to the laboratory at 38.50° C.

[0275] Egg Stripping:

[0276] 1—Pick up oocytes with a pipette and place in ˜1 ml ofhyaluronidase (1 mgr in HTF-BSA) solution in a 15 ml conical tube.

[0277] 2—Vortex for 5 minutes at a setting of 6. Pour ˜5 ml HTF-BSA intoconical tube containing oocytes.

[0278] 3—Swirl and pour into a 60 mm bacterial plate. Repeat rinse andplace plate on a dissecting scope.

[0279] 4—Allow oocytes to settle.

[0280] 5—Swirl plate to center oocytes.

[0281] 6—Pick up all oocytes and place in a second dish of HTF-BSA.

[0282] 7—Center oocytes again, pick up with a pipette and place in adish of M199+10% fetal calf serum (FCS). Incubate at 38.5°0 C. and 5%CO₂ in air until use.

[0283] Oocyte Enucleation:

[0284] 1—Pipette 2 to 4, 100-200 μl, drops of manipulation media(HTF-BSA) onto a 100 mm bacterial plate. Use the pipette tip to spreadout and flatten the drops.

[0285] 2—Pipette enough mineral oil on top of the drops until they arecovered evenly.

[0286] 3—Place the plate onto the stage of the inverted scope. Positionone drop directly over the middle of the stage. Set the magnification atits lowest setting.

[0287] 4—Fill the tubing between the manipulators and the opening forthe manipulation needles with water. Be sure there are no air bubbles inthe tubing.

[0288] 5—Clean the needles: Make a 5 ml syringe with a blunted 18Gneedle. Connect this to a short piece of the same tubing as used formanipulation. Use this to aspirate glass-cleaning solution through oneglass manipulation needle. Then place the needle tip into water that hasbeen brought to a boil. Aspirate multiple drops of hot water to rinseout the cleaning solution and any remaining debris. Repeat with theother glass needle. The rinsing apparatus can be saved and usedrepeatedly.

[0289] 6—Fill a cleaned, rinsed enucleation needle and holding needlewith Flourinert oil. Be sure there are no air bubbles and that theneedles are filled to the very end. To do this, fill a 3 ml syringe withFlourinert. Attach a short piece of the same tubing as used formanipulation. Connect the tubing to the blunt end of the glass needleand gently depress the plunger to fill the needle. Repeat for the otherglass needle.

[0290] 7—One at a time, attach each needle to the filled tubing,preventing air between the connections. Position each needle over theplate, approximately over the middle of the stage, and fasten to themanipulator arms.

[0291] 8—Lower the needles into a drop on the manipulation plate. Lookthrough the eyepieces to center and fine tune the needles by rotatingthe needles into the position you prefer. Advance the meniscus ofFlourinert oil until it can be viewed under higher power and movessmoothly up and down the needles.

[0292] 9—After setting up, rotate the plate to move the needles into anew clean drop of media.

[0293] 10—The stained oocytes are removed and placed in close proximityto the enucleation tools in the manipulation plate. This is generallydone by having the tools, positioned and in focus, at 40× on theinverted scope and then, while monitoring through the objective, using apipette to deposit the oocytes near the tips of the tools.

[0294] 11—For enucleation, the oocytes are visualized using an invertedmicroscope with an HMC objective at 200× magnification and an UVlighting system. The UV lighting system should include a UniBlitzshutter and foot pedal to shorten the length of time oocytes are exposedto UV light.

[0295] 12—Each oocyte is held with the holding pipette and rotated usingthe enucleation needle until the metaphase plate can clearly be seen onthe margin of the cytoplasm. The enucleation pipette is then insertedthrough the zona pellucida to a position near the chromosomes and themetaphase plate is aspirated along with a minimum of cytoplasm. Thetotal volume of cytoplasm should not exceed the length of the bevel ofthe enucleation needle.

[0296] 13—UV lighting is damaging to oocytes and should only be used fora minimum of time. Oocytes should be positioned using only brief flashesof UV.

[0297] 14—Successful enucleation is confirmed when the enucleationpipette is removed from the oocyte with the chromosomes visible insidethe pipette tip.

[0298] 15—Completed groups of oocytes are transferred to a well ofM199+FCS at 37° C. and 5% CO₂ in air until cell transfer.

[0299] Variations:

[0300] A. The rabbit oocyte cytoplasm may be transferred to anotheroocyte; e.g., of a third species. Including but not limited to Bostaurus, Bos indicus, and Bos gauurus. Bos gaurus provides an advantagein that this third species has no bovine spongiform encephalopathy(prion disease). The effect of this step is to “spike” the third speciesoocyte with rabbit ooplasm.

[0301] B. The rabbit oocyte may be genetically modified using a zonapellucida-specific promoter driving a gene of interest, or may bemodified by some other means; i.e., by injecting proteins, in order toimprove reprogramming efficency or to limit the differentiationpotential of the embryo, or for other desired purposes.

[0302] Cell Transfer:

[0303] 1—Remove media from human fibroblast cell line. Rinse cells withDPBS, overlay with a minimum amount of pronase solution and place on awarming plate.

[0304] 2—When cells are loose (generally a few minutes), pipette toseparate into a single cell suspension and transfer to a conical tube.Add 5-10 ml of M199+FCS to stop the enzymatic action of the pronase.

[0305] 3—Spin the tube in the centrifuge to pellet the cells.

[0306] 4—Aspirate the supernatant and re-suspend the cell pellet in asmall amount of HTF-BSA. The amount will depend on the pellet size andconcentration desired.

[0307] 5—Transfer 5-20 μl of cell suspension to one drop on themanipulation plate.

[0308] 6—20-40 enucleated oocytes are picked up and placed in the samemanipulation drop as the cells. Alternatively, use separate drops forcells and oocytes, and move back and forth between the two drops.

[0309] 7—Enucleated oocytes and cells are viewed at 200× on the invertedscope.

[0310] 8—Individual cells are selected and picked up using the transferpipette until there are 1-20 cells in the pipette.

[0311] 9—Enucleated oocytes are held with the holding pipette androtated with the tip of the transfer pipette, until the polar body isvisible in the space closest to the tip of the transfer needle. Thetransfer pipette is inserted through the zona pellucida and one cell iscarefully placed in the perivitelline space, next to the polar body ifpossible. Placement of the cell next to the polar body helps duringalignment for fusion but in some cases the polar body is not present andthe cell is placed anywhere in the perivitteline space.

[0312] 10-When each group of oocytes is finished it is placed back inM199+FCS media at 37° C. and 5% CO₂ in air until fusion.

[0313] Fusion and Activation:

[0314] 1—Procedure starts at 17-18 hrs after superovulation

[0315] 2—Fusion is simultaneous with the first set of activation

[0316] 3—Using sterile forceps, remove the fusion chamber from EtOH andimmerse it in a 50 ml conical tube of sterile DBPS, rinse carefully.Transfer to a second tube of DBPS until use.

[0317] 4—Place two small portions of vacuum grease ˜9 cm apart on theinside surface of a 100 mm bacterial plate. Remove the fusion chamberfrom the DBPS and place on the plate with both ends on the vacuumgrease. Press firmly into place. Flood plate with fusion media, beingsure to cover chamber completely.

[0318] 5—Make a manipulation tip:

[0319] a) Pull a glass pipette on the micropipette puller using program#2 (see instruction manual for specific directions).

[0320] b) Attach the pipette to the microforge in a vertical position.

[0321] c) Carefully melt the tip of the pipette until it is completelyclosed and has a round tip.

[0322] 6—Place the chamber on top of the stage warmer in the 2nddissecting scope. The fusion machine is hooked up to the chamber withthe black lead (−) attached the upper electrode and the red lead (+)attached to the lower electrode.

[0323] 7—The fusion machine is set for of 3 DC pulses of 250 Kv/cm for20 μsec. Several test pulses should be tried before NTs are fused.

[0324] 8—NT units are removed from M199+FCS media and placed in HTF-BSA

[0325] 9—Place eggs in a place with Mannitol media (0.3 mM) and let themsettle to the bottom of the plate before moving to the fusion/activationchamber.

[0326] 10—A small number of NT units are picked up (usually 4-8) fromthe group and placed between the electrodes in the fusion chamber. Themanipulation tip is used to spin each embryo until they are all alignedwith the transferred cell closest to the negative electrode and theoocyte closest to the positive electrode. A pulse is given, the embryosare removed from the fusion chamber, and are placed in M199+FCS at 37°C., 5% CO₂. Alternatively, solution of from 37° C. to 38.5° C. can beused.

[0327] 11—One hour later steps 8, 9 and 10 are repeated.

[0328] 12—After the second set of stimulation they were incubated inSpgr Cycloheximide and 2 mM DMAP in M199 for one hour then,

[0329] 13—washed and cultured in M199+10% FCS

[0330] Embryo Culture:

[0331] 1—Preparing culture plates: warmed M199 media with 10% FBS/well(alternatively, rabbit vitreous humor can be used). Overlay each wellwith enough mineral oil to cover media. Be sure that the mineral oil hasbeen filtered through at least a 0.45 μm filter before use.

[0332] 2—Prepared culture plates should equilibrate at 37° C. and 5% CO₂in air for at least 3 hours before use. It is preferable to equilibratethem overnight.

[0333] Alternatively, the plates can be equilibrated at from 37° C. to38.5° C.

[0334] 3—Remove embryos from the activation plates. Rinse 3× in HTF-BSAand place in the prepared culture plates. This is considered Day 0 ofembryo development. It is preferable to place less than 50 embryos perwell.

[0335] 4—Incubate throughout culture at 37° C. and 5% CO₂ in air.Alternatively, the embryos can be incubated at from 37° C. to 38.5° C.

[0336] 5—On Day 3 transfer the embryos to a fresh culture plate(prepared as above). Only the cleaved embryos should be moved. Thepercent cleavage should be determined and recorded at this time.

[0337] 6—Evaluate for blastocyst development on days 5, 6 and 7 ofculture.

[0338] 7—Blastocysts should be subjected to immunosurgery.

[0339] 8—All embryos that do not develop after day 13 must be recordedand discarded.

[0340] Immunosurgery:

[0341] 1—Make 3-20 μl drops of pronase (protease) under oil and move theblastocysts from drop 1 to 3. Leave them in drop 3 and wait until thezona pellucida is dissolved. Look at the blastocyst constantly and assoon as the zona disappear they must be removed.

[0342] 2—Rinse 6 times in HTF-BSA.

[0343] 3—Make 3-20 μl drops of antibody (Sigma polyclonal against wholehuman serum albumin H-8765 diluted 1:3 in G2) under oil and move theblastocysts from drop 1 to 3. Leave them in drop 3 for 30 minutes.

[0344] 4—Rinse 6 times in HTF-BSA

[0345] 5—Make 3-20 μl drops of Guinea Pig complement diluted 1:3 in G2under oil and move the blastocysts from drop 1 to 3. Leave them in drop3 for 30 minutes. The blastocyst should collapse.

[0346] 6—Rinse 6 times in HTF-BSA

[0347] 7—Place the remaining of the embryo (ICM) in mitoticallyinactivated mouse feeder layer and culture with DMEM+15% FCS.

Results

[0348] Nuclear transfer was performed as described above, using somaticnuclear donor cells from a 2 year-old human donor. As of Feb. 28, 2002,six out of forty one nuclear transfer embryos had developed to themorula stage, and one was compacting, a sign of genome activation.

What is claimed is:
 1. A method of producing embryonic or stem-likecells comprising the following steps: (i) inserting a desireddifferentiated human or mammalian cell or cell nucleus into anenucleated animal oocyte, wherein such oocyte is derived from adifferent animal species than the human or mammalian cell underconditions suitable for the formation of a nuclear transfer (NT) unit;(ii) activating the resultant nuclear transfer unit; (iii) culturingsaid activated nuclear transfer unit until greater than the 2-celldevelopmental stage; and (iv) culturing cells obtained from saidcultured NT units to obtain embryonic or stem-like cells.
 2. The methodof claim 1, wherein the cell inserted into the enucleated animal oocyteis a human cell.
 3. The method of claim 2, wherein said human cell is anadult cell.
 4. The method of claim 2, wherein said human cell is anepithelial cell, keratinocyte, lymphocyte or fibroblast.
 5. The methodof claim 2, wherein the oocytes are obtained from a mammal.
 6. Themethod of claim 5, wherein the animal oocyte is obtained from anungulate.
 7. The method of claim 6, wherein said ungulate is selectedfrom the group consisting of bovine, ovine, porcine, equine, caprine,and buffalo.
 8. The method of claim 1, wherein the enucleated oocyte ismatured priorto enucleation.
 9. The method of claim 1, wherein the fusednuclear transfer units are activated in vitro.
 10. The method of claim1, wherein the activated nuclear transfer units are cultured on a feederlayer culture.
 11. The method of claim 10, wherein the feeder layercomprises fibroblasts.
 12. The method of claim 1, wherein in step (iv)cells from a NT unit having 16 cells or more are cultured on a feedercell layer.
 13. The method of claim 12, wherein said feeder cell layercomprises fibroblasts.
 14. The method of claim 13, wherein saidfibroblasts comprise mouse embryonic fibroblasts.
 15. The method ofclaim 1, wherein the resultant embryonic or stem-like cells are inducedto differentiate.
 16. The method of claim 2, wherein the resultantembryonic or stem-like cells are induced to differentiate.
 17. Themethod of claim 1, wherein fusion is effected by electrofusion. 18.Embryonic or stem-like cells obtained according to the method ofclaim
 1. 19. Human embryonic or stem-like cells obtained according tothe method of claim
 2. 20. Human embryonic or stem-like cells obtainedaccording to the method of claim
 3. 21. Human embryonic or stem-likecells obtained according to the method of claim
 4. 22. Human embryonicor stem-like cells obtained according to the method of claim
 6. 23.Human embryonic or stem-like cells obtained according to the method ofclaim
 7. 24. Differentiated human cells obtained by the method of claim16.
 25. The differentiated human cells of claim 24, which are selectedfrom the group consisting of neural cells, hematopoietic cells,pancreatic cells, muscle cells, cartilage cells, urinary cells, livercells, spleen cells, reproductive cells, skin cells, intestinal cells,and stomach cells.
 26. A method of therapy which comprises administeringto a patient in need of cell transplantation therapy isogenicdifferentiated human cells according to claim
 24. 27. The method ofclaim 26, wherein said cell transplantation therapy is effected to treata disease or condition selected from the group consisting of Parkinson'sdisease, Huntington's disease, Alzheimer's disease, ALS, spinal corddefects or injuries, multiple sclerosis, muscular dystrophy, cysticfibrosis, liver disease, diabetes, heart disease, cartilage defects orinjuries, burns, foot ulcers, vascular disease, urinary tract disease,AIDS and cancer.
 28. The method of claim 26, wherein the differentiatedhuman cells are hematopoietic cells or neural cells.
 29. The method ofclaim 26, wherein the therapy is for treatment of Parkinson's diseaseand the differentiated cells are neural cells.
 30. The method of claim26, wherein the therapy is for the treatment of cancer and thedifferentiated cells are hematopoietic cells.
 31. The differentiatedhuman cells of claim 24, which contain and express an inserted gene. 32.The method of claim 1, wherein a desired gene is inserted, removed ormodified in said embryonic or stem-like cells.
 33. The method of claim32, wherein the desired gene encodes a therapeutic enzyme, a growthfactor or a cytokine.
 34. The method of claim 32, wherein said embryonicor stem-like cells are human embryonic or stem-like cells.
 35. Themethod of claim 32, wherein the desired gene is removed, modified ordeleted by homologous recombination.
 36. The method of claim 1, whereinthe donor cell is genetically modified to impair the development of atleast one of endoderm, ectoderm and mesoderm.
 37. The method of claim 1,wherein the donor cell is genetically modified to increasedifferentiation efficiency.
 38. The method of claim 36, wherein thecultured nuclear transfer unit is cultured in a media containing atleast one capsase inhibitor.
 39. The method of claim 1, wherein thedonor cell expresses a detectable label that is indicative of theexpression of a particular cyclin.
 40. The method of claim 36, whereinthe donor cell has been modified to alter the expression of a geneselected from the group consisting of SRF, MESP-1, HNF4, beta-1,integrin, MSD, GATA-6, GATA-4, RNA helicase A, and H beta
 58. 41. Themethod of claim 37, wherein said donor cell has been geneticallymodified to introduce a DNA that provides for expression of the Q7and/or Q9 genes.
 42. The method of claim 41, wherein said gene or genesare operably linked to a regulatable promoter.
 43. The method of claim1, wherein the donor cell has been genetically modified to inhibitapoptosis.
 44. The method of claim 43, wherein reduced apoptosis isprovided by altering expression of one or more genes selected from thegroup consisting of Bad, Bok, BH3, Bik, BIk, Hrk, BNIP3, GimL, Bid,EGL-1, Bcl-XL, Bcl-w, McI-1, A1, Nr-13, BHRF-1, LMW5-HL, ORF16,Ks-Bcl-2, E1B-19K, and CED-9.
 45. The method of claim 44, wherein atleast one of said genes is operably linked to an inducible promoter. 46.A mammalian somatic cell that expresses a DNA that encodes a detectablemarker, the expression of which is linked to a particular cyclin. 47.The cell of claim 46, wherein the cyclin is selected from the groupconsisting of cyclin D1, D2, D3, B1, B2, E, A and H.
 48. The cell ofclaim 46, wherein the detectable marker is a fluorescent polypeptide.49. The cell of claim 48, wherein said mammalian cell is selected fromthe group consisting of human, primate, rodent, ungulate, canine, andfeline cells.
 50. The cell of claim 48, wherein said cell is a human,bovine or primate cell.
 51. A method of producing human embryonicstem-like cells, comprising: (i) inserting a human cell or cell nucleusinto a recipient oocyte that is derived from a non-human mammal, underconditions suitable for the formation of a nuclear transfer (NT) unit;(ii) activating the resultant NT unit; (iii) culturing the activated NTunit until greater than the 2-cell developmental stage; and (iv)culturing cells obtained from the cultured NT unit to obtain humanembryonic stem-like cells.
 52. The method of claim 51, wherein therecipient oocyte is enucleated prior to inserting the human cell or cellnucleus.
 53. The method of claim 51, wherein the oocyte comprises aprotein or other substance that improves reprogramming efficiency orlimits the differentiation potential of the NT unit.
 54. The method ofclaim 52, wherein the recipient oocyte is a rabbit oocyte.
 55. Themethod of claim 54, wherein the recipient oocyte is spiked with rabbitooplasm.
 56. The method of claim 55, wherein the oocyte comprises aprotein or other substance that improves reprogramming efficiency orlimits the differentiation potential of the nuclear transfer unit. 57.The method of claim 51, wherein the activated NT unit is cultured untilthe blastocyst stage.
 58. A pluripotent human embryonic stem-like cellmade by the method of claim
 51. 59. The pluripotent human embryonicstem-like cell of claim 58, which cell contains mitochondrial DNA of therecipient oocyte.
 60. A pluripotent human embryonic stem-like cell madeby the method of claim
 54. 61. The pluripotent human embryonic stem-likecell of claim 60, which cell contains rabbit mitochondrial DNA.
 62. Amethod for producing a nuclear transfer human embryo by cross-speciesnuclear transfer comprising: (i) transferring a human cell or humannucleus into a rabbit oocyte which is enucleated before, simultaneous orafter said human cell or nuclear transfer; and (ii) the resultant fusionis permitted to develop into a nuclear transfer embryo.
 63. The methodof claim 62 which comprises transferring the pronucleus that resultsafter step (ii) into another oocyte which is enucleated before,simultaneous or after transfer.
 64. The method of claim 62 which furthercomprises transferring additional ooplasm from another rabbit oocyteinto the resultant fusion.
 65. The method of claim 62 wherein theresultant fusion is activated simultaneous to fusion to induce embryonicdevelopment.
 66. The method of claim 62 wherein the rabbit oocyte isactivated prior to transferral of said human cell or nucleus therein.67. The method of claim 62 which includes an activation step effectedafter said human cell or human nuclear transfer.
 68. The method of claim65, 66 or 67 wherein activation includes the use of DMAP andcycloheximide.
 69. The method of claim 62 wherein the nuclear transferembryo produced by step (ii) is cultured on a feeder layer.
 70. Themethod of claim 69 wherein said culture gives rises to human pluripotentcells.
 71. The method of claim 70 wherein said pluripotent cells arepermitted to differentiate into different cell types.
 72. The method ofclaim 69 wherein the embryo is cultured on a fibroblast feeder layer.73. A method for enhancing the development of a cross-species nucleartransfer unit produced-by transferral of a human cell or nucleus into anon-human oocyte, comprising introducing into said oocyte or said humancell prior to transferral ooplasm from a rabbit oocyte.
 74. The methodof claim 73 wherein said non-human oocyte is an ungulate oocyte.
 75. Themethod of claim 74 wherein said ungulate oocyte is a bovine oocyte. 76.A cross-species blastula or morula that results from the process ofclaim 62 or claim
 73. 77. Differentiated cells derived from the morulaor blastocyst of claim 76.